WO2001007610A1

WO2001007610A1 - Pmf-1 (polyamide-modulated factor 1)

Info

Publication number: WO2001007610A1
Application number: PCT/US2000/019994
Authority: WO
Inventors: Robert A. Casero, Jr.; Yanlin Wang; Anthony E. Pegg
Original assignee: Johns Hopkins University School Of Medicine
Priority date: 1999-07-23
Filing date: 2000-07-21
Publication date: 2001-02-01
Also published as: AU6365700A

Abstract

An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding human polyamine-modulated factor-1 (PMF-1), a variant PMF-1, or a fragment of either of the foregoing, an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding human PMF-1, a variant PMF-1 or a fragment of either of the foregoing, a vector comprising such an isolated or purified nucleic acid molecule, a cell comprising such a vector, an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding human PMF-1, a variant PMF-1 or a fragment of either of the foregoing, a conjugate comprising such an isolated or purified polypeptide molecule and a targeting moiety, a hybridoma cell line that produces a monoclonal antibody that is specific for an aforementioned isolated or purified polypeptide molecule, the monoclonal antibody produced by the hybridoma cell line, a polyclonal antiserum raised against an aforementioned isolated or purified polypeptide molecule, a method of regulating a PMF-1-responsive, polyamine-dependent gene in a cell, a method of assessing the sensivity of a cell to treatment with a polyamine or an analogue thereof, and a method of assessing the effectiveness of treatment of a cell with a polyamine or an analogue thereof.

Description

PMF-1 (POLYAMIDE-MODULATED FACTOR 1)

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made in part with Government support under Grant Number CA 51085 awarded by the National Cancer Institute, which is part of the National Institutes of Health. Therefore, the Government may have certain rights in this invention.

TECHNICAL FIELD OF THE INVENTION The present invention relates to an isolated or purified nucleic acid molecule, a vector comprising such a nucleic acid molecule, a cell comprising such a vector, an isolated or purified polypeptide, a conjugate comprising such a polypeptide and a targeting moiety, a hybridoma cell line, a monoclonal antibody, a polyclonal antiserum, and related methods of use.

BACKGROUND OF THE INVENTION The absolute requirement for polyamines in the growth of eukaryotic cells has led to the targeting of their metabolic pathway as a means of antineoplastic intervention (Marton et al., Annu. Rev. Pharmacol. 35: 55-91 (1995)). Several newly synthesized polyamine analogues designed to alter regulation of polyamine metabolism are currently under investigation for their antitumor activity (Saab et al., J. Med. Chem. 36: 2998-3004 (1993); Casero et al, Cancer Chemother. Pharmacol. 36: 69-74 (1995); Bernacki et al, Clin. Cancer Res. 1 : 847-857 (1995); and Bergeron et al., J. Med. Chem. 40: 1475-1494 (1997)). Some of these analogues appear to exerttheir cytotoxic effects in association with the superinduction of spermidine/spermine N¹- acetyltransferase (SSAT; see, e.g., U.S. Patent No. 5,840,559), the rate-limiting step of polyamine catabolism (Casero et al., Cancer Res. 49: 3829-3833 (1989); Casero et al., Cancer Res. 52: 5359-5363 (1992); Casero et al, Cancer Res. 54: 3955-3958 (1994); Alhonen et al, J. Biol. Chem. 273: 1964-1969 (1998); Porter et al., Cancer Res. 51 : 3715-3720 (1991); Porter et al, Cancer Res. 53: 581-586 (1993); and Ha et al., Proc. Natl. Acad. Sci. U. S. A. 94: 11557-11562 (1997)). The initial induction of this enzyme occurs at the level of increased transcription in response to analogue treatment (Fogel- Petrovic et al., J. Biol. Chem. 268: 19118-19125 (1993); Xiao et al, Biochem. J. 313: 691-696 (1996); and Wang et al., J. Biol. Chem. 273: 34623-34630 (1998)). A xacting polyamine-responsive element (PRE) and a trans- acting protein, the transcription factor Nrf-2, recently have been identified as involved in the regulation of SSAT gene transcription (Wang et al. (1998), supra). However, the Nrf-2 transcription factor appears to be constitutively expressed only in those tumor cell types capable of expressing SSAT at high levels. Furthermore, the binding of Nrf-2 to the PRE does not change in response to treatment with a natural polyamine or an analogue thereof as measured by electrophoretic mobility shift assays (Wang et al. (1998), supra). The results of these recent studies suggest at least two possibilities: 1) PRE-bound Nrf-2 is altered by analogue treatment, leading to transcriptional activation, or 2) an additional factor that is induced by analogue exposure leads to transcriptional activation of the SSAT gene.

In view of the above, it is an object of the present invention to provide an additional human factor which is induced in response to a polyamine or an analogue thereof, interacts with the leucine zipper region of Nrf-2, and, together with Nrf-2, activates SSAT transcription. This and other objects and advantages of the present invention, as well as additional inventive features, will become apparent upon reading the detailed description of the invention provided herein. BRIEF DESCRIPTION OF THE INVENTION The present invention provides an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding human polyamme-modulated factor- 1 (PMF-1) or a fragment thereof The isolated or punfied nucleic acid molecule can (I) encode the ammo acid sequence of SEQ ID NO: 2 or a fragment thereof such that the isolated or purified nucleic acid molecule comprises at least 17 nucleotides, (n) consist essentially of the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof compnsmg at least 17 nucleotides, (in) hybndize under stnngent conditions to an isolated or purified nucleic acid molecule consisting essentially of the nucleotide sequence that is complementary to SEQ ID NO: 1 or a fragment thereof, or (iv) share 90% or more identity with SEQ ID NO. 1. Also provided is an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a vanant PMF-1 or a fragment thereof compnsmg at least 17 nucleotides.

The present invention also provides an isolated or punfied nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding human PMF-1 or a fragment thereof. The isolated or punfied nucleic acid molecule can (l) be complementary to a nucleotide sequence encoding the ammo acid sequence of SEQ ID NO: 2 or a fragment thereof such that the isolated or punfied nucleic acid molecule compnses at least 17 nucleotides, (n) be complementary to the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof compnsing at least 17 nucleotides, (in) hybndize under stringent conditions to an isolated or punfied nucleic acid molecule consisting essentially of SEQ ID NO: 1 or a fragment thereof, or (iv) share 90% or more identity with the nucleotide sequence that is complementary to SEQ ID NO: 1 or a fragment thereof. Also provided is an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to either of a nucleotide sequence encoding a vanant PMF-1 or a fragment thereof comprising at least 17 nucleotides.

The present invention further provides an isolated or punfied DNA molecule consisting essentially of the genomic sequence of human PMF-1 or a fragment thereof. The isolated or punfied DNA molecule can consist essentially of the nucleotide sequence of SEQ ID NO: 3 or a fragment thereof of at least 16 nucleotides. 4 Also provided by the present invention is a vector comprising one of the above- described isolated or purified nucleic acid molecules. Further provided is a cell comprising such a vector.

An isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding human PMF-1 or a fragment thereof, either one of which is optionally glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated or converted into an acid addition salt, is also provided by the present invention. The isolated or purified polypeptide molecule can (i) consist essentially of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof comprising at least 8 amino acids or (ii) share 90% or more identity with SEQ ID NO: 2 or a fragment thereof. Also provided is an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a variant PMF-1 or a fragment thereof comprising at least 8 amino acids, which is optionally glycoslyated, amidated, carboxylated, phosphorylated, esterified, N-acylated or converted into an acid addition salt. Further provided is a conjugate comprising an above-described isolated or purified polypeptide molecule and a targeting moiety. The targeting moiety can be an antibody or an antigenically reactive fragment thereof.

Still further provided is a hybridoma cell line that produces a monoclonal antibody that is specific for an above-described isolated or purified polypeptide molecule. Thus, the monoclonal antibody produced by the hybridoma cell line is also provided as is a polyclonal antiserum raised against an above-described isolated or purified polypeptide molecule.

Yet still further provided is a method of regulating a PMF-1 -responsive gene in a cell. The method comprises contacting the cell with a gene-regulating amount of PMF-1, whereupon the PMF-1 -responsive gene in the cell is regulated. The PMF-1 - responsive gene preferably encodes spermidine/spermine N'-acetyltransferase (SSAT). Desirably, PMF-1 regulates the PMF-1 -responsive gene by increasing the transcription of the gene and by super-inducing the gene. In one embodiment of the method, the cell is contacted with PMF-1 by contacting the cell with an above-described isolated or purified nucleic acid molecule which consists essentially of a nucleotide sequence which encodes PMF- 1 or a functional fragment thereof and which is operably linked to a promoter, wherein the isolated or punfied nucleic acid molecule is optionally in the form of a vector In another embodiment of the method, the cell is contacted with PMF-1 by contacting the cell with an above-described isolated or punfied polypeptide molecule consisting essentially of an ammo acid sequence encoding PMF-1 or a fragment thereof The isolated or punfied polypeptide molecule can be contained within a hposome compnsmg a cell-surface targeting moiety that binds to the cell being contacted In yet another embodiment, the cell is contacted with PMF-1 by contacting the cell with an above-described conjugate. The cell can be a cancerous cell and the method decreases the viability and/or metastatic potential of the cancerous cell When the cell is a cancerous cell, the method can further comprise the administration of an anti-cancer agent

Also still further provided is a method of assessing the sensitivity of cells to treatment with a polyamine or an analogue thereof. The method compnses assessing the level of PMF-1 mRNA or PMF-1 polypeptide m a sample of the cells after contact with a polyamine or an analogue thereof, wherein an increase m the level of PMF-1 mRNA or PMF- 1 polypeptide in the sample of cells after contact with a polyamine or an analogue thereof as compared to a control sample or a sample of the cells before contact with the polyamine or analogue thereof is indicative of sensitivity of the cells to treatment with a polyamine or an analogue thereof. The cell can be a cancerous cell. A method of assessing the effectiveness of treatment of cells with a polyamine or an analogue thereof is also provided. The method comprises assessing the level of PMF-1 mRNA or PMF-1 polypeptide in a sample of the cells before and dunng treatment of the cells with a polyamine or an analogue thereof, wherein an increase m the level of PMF-1 mRNA or PMF-1 polypeptide in the cells dunng treatment of the cells with a polyamine or an analogue thereof is indicative of efficacy of treatment of the cells with a polyamine or an analogue thereof

BRIEF DESCRIPTION OF THE FIGURES Fig 1 represents the nucleotide (SEQ ID NO: 1) and deduced ammo acid (SEQ ID NO: 2) sequences of human PMF-1 cDNA.

Fig. 2 represents the genomic sequence (SEQ ID NO: 3) of human PMF-1. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding human polyamine-modulated factor- 1 (PMF- 1 ) or a fragment thereof. By "isolated" is meant the removal of a nucleic acid from its natural environment. By "purified" is meant that a given nucleic acid, whether one that has been removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and/or amplified under laboratory conditions, has been increased in purity, wherein "purity" is a relative term, not "absolute purity." "Nucleic acid molecule" is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides.

Preferably, the isolated or purified nucleic acid molecule that consists essentially of a nucleotide sequence encoding human PMF- 1 or a fragment thereof (i) encodes the amino acid sequence of SEQ ID NO: 2 or a fragment thereof such that the isolated or purified nucleic acid molecule comprises at least 17 nucleotides, (ii) consists essentially of the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof, (iii) hybridizes under stringent conditions to an isolated or purified nucleic acid molecule consisting essentially of the nucleotide sequence that is complementary to SEQ ID NO: 1 or a fragment thereof, or (iv) shares 90% or more identity with SEQ ID NO: 1. SEQ ID NOS: 1 and 2 are also available as accession no. AF141310 through the NCBI database at www.ncbi.nlm.nih.gov.

Also provided is an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a variant PMF-1 or a fragment thereof comprising at least 17 nucleotides. The variant comprises one or more insertions, deletions and/or substitutions. Desirably, the variant PMF-1 does not differ functionally from the corresponding unmodified PMF-1, such as that comprising SEQ ID NO: 1. Preferably, the variant PMF-1 increases transcription of the gene encoding SSAT at least about 50%, more preferably at least about 75%, most preferably at least about 90%) as well as the corresponding unmodified PMF-1 as determined by in vitro assay in the presence of excess Nrf-2. The manner in which the assay is carried out is not critical and can be conducted in accordance with methods known in the art. Preferably, the one or more substitution(s) do(es) not result in a change in an amino acid of the encoded PMF-1 or results in the substitution of an amino acid of the encoded PMF-1 with another amino acid of approximately equivalent size, shape and charge. The present invention also provides an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding human PMF-1 or a fragment thereof. Such an isolated or purified nucleic acid molecule preferably (i) is complementary to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 or a fragment thereof such that the isolated or purified nucleic acid molecule comprises at least 17 nucleotides, (ii) is complementary to the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof comprising at least 17 nucleotides, (iii) hybridizes under stringent conditions to an isolated or purified nucleic acid molecule consisting essentially of SEQ ID NO: 1 or a fragment thereof, or (iv) shares 90% or more identity with the nucleotide sequence that is complementary to SEQ ID NO: 1 or a fragment thereof. Also provided is an isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to either of a nucleotide sequence encoding a variant PMF- 1 or a fragment thereof comprising at least 17 nucleotides as described above. The present invention further provides an isolated or purified DNA molecule consisting essentially of the genomic sequence of human PMF-1 or a fragment thereof. The isolated or purified DNA molecule can consist essentially of the nucleotide sequence of SEQ ID NO: 3 or a fragment thereof of at least 16 nucleotides. SEQ ID NO: 3 is also available as accession no. AH008078 through the NCBI database at the web site indicated above.

With respect to the above, one of ordinary skill in the art knows how to generate insertions, deletions and/or substitutions in a given nucleic acid molecule. See, for example, the references cited herein under "Examples." With respect to the above isolated or purified nucleic acid molecules, it is preferred that any such insertions, deletions and/or substitutions are introduced into the 5' region of the nucleotide sequence encoding PMF-1 or a variant thereof and that no insertions, deletions and or substitutions are introduced into the 3 ' region, particularly the coiled- coil region, of the nucleotide sequence encoding PMF-1 or a variant thereof. It is also preferred that the one or more substitution(s) do(es) not result in a change in an amino acid of PMF-1. Alternatively, and also preferred, is that the one or more substitution(s) result(s) in the substitution of an amino acid with another amino acid of approximately equivalent size, shape and charge.

Also with respect to the above, "does not differ functionally from" is intended to mean that the variant PMF-1 has activity characteristic of the unmodified PMF-1. In other words, it regulates a PMF-1 -responsive gene. However, the variant PMF-1 can be more or less active than the unmodified PMF-1 as desired in accordance with the present invention.

An indication that polynucleotide sequences are substantially identical is if two molecules selectively hybridize to each other under stringent conditions. The phrase "hybridizes to" refers to the selective binding of a single-stranded nucleic acid probe to a single-stranded target DNA or RNA sequence of complementary sequence when the target sequence is present in a preparation of heterogeneous DNA and/or RNA. "Stringent conditions" are sequence-dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.

For example, under stringent conditions, as that term is understood by one skilled in the art, hybridization is preferably carried out using a standard hybridization buffer at a temperature ranging from about 50°C to about 75°C, even more preferably from about 60°C to about 70°C, and optimally from about 65°C to about 68°C. Alternately, formamide can be included in the hybridization reaction, and the temperature of hybridization can be reduced to preferably from about 35°C to about 45°C, even more preferably from about 40°C to about 45°C, and optimally to about 42°C. Desirably, formamide is included in the hybridization reaction at a concentration of from about 30% to about 50%, preferably from about 35% to about 45%, and optimally at about 40%. Moreover, optionally, the hybridized sequences are washed (if necessary to reduce non- specific binding) under relatively highly stringent conditions, as that term is understood by those skilled m the art For instance, desirably, the hybndized sequences are washed one or more times using a solution compnsing salt and detergent, preferably at a temperature of from about 50°C to about 75°C, even more preferably at from about 60°C to about 70°C, and optimally from about 65°C to about 68°C. Preferably, a salt (e.g., such as sodium chlonde) is included in the wash solution at a concentration of from about 0.01 M to about 1 0 M Optimally, a detergent (e.g., such as sodium dodecyl sulfate) is also included at a concentration of from about 0.01% to about 1.0%.

In view of the above, "stringent conditions" preferably allow for from about 25% to about 5% mismatch, more preferably from about 15% to about 5% mismatch, and most preferably from about 10% to about 5% mismatch. "At least moderately stringent conditions" preferably allow for from about 40% to about 15% mismatch, more preferably from about 30% to about 15% mismatch, and most preferably from about 20% to about 15% mismatch. "Low stringency conditions" preferably allow for from about 60% to about 35% mismatch, more preferably from about 50% to about 35% mismatch, and most preferably from about 40% to about 35% mismatch. With respect to the preceding ranges of mismatch, 1% mismatch corresponds to one degree decrease m the melting temperature

The above isolated or purified nucleic acid molecules also can be charactenzed m terms of "percentage of sequence identity " In this regard, a given nucleic acid molecule as described above can be compared to a nucleic acid molecule encoding a corresponding gene (i.e., the reference sequence) by optimally aligning the nucleic acid sequences over a comparison window, wherein the portion of the polynucleotide sequence m the comparison window may compnse additions or deletions (i.e., gaps) as compared to the reference sequence, which does not compnse additions or deletions, for optimal alignment of the two sequences. The percentage of sequence identity is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences, i.e., the number of matched positions, dividing the number of matched positions by the total number of positions m the window of companson, and multiplying the result by 100 to yield the percentage of sequence identity Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algonthms (e.g., GAP, BESTFIT, FASTA, and TFASTA m the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI, or BlastN and BlastX available from the National Center for Biotechnology Information, Bethesda, MD), or by inspection Sequences are typically compared using BESTFIT or BlastN with default parameters "Substantial sequence identity" means that at least 75%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the sequence of a given nucleic acid molecule is identical to a given reference sequence. Typically, two polypeptides are considered to be substantially similar if at least 40%, preferably at least 60%, more preferably at least 90%, and most preferably at least 95% of the ammo acids of which the polypeptides are comprised are identical to or represent conservative substitutions of the ammo acids of a given reference sequence

One of ordinary skill m the art will appreciate, however, that two polynucleotide sequences can be substantially different at the nucleic acid level, yet encode substantially similar, if not identical, ammo acid sequences, due to the degeneracy of the genetic code The present invention is intended to encompass such polynucleotide sequences

While the above-described nucleic acid molecules can be isolated or purified, alternatively they can be synthesized. Methods of nucleic acid synthesis are known m the art See, e.g , the references cited herein under "Examples "

The above-described nucleic acid molecules can be used, m whole or m part (i.e , as fragments or primers), to identify and isolate corresponding genes from other organisms for use m the context of the present inventive method using conventional means known m the art See, for example, the references cited herein under "Examples "

In view of the above, the present invention also provides a vector compnsing an above-described isolated or purified nucleic acid molecule. A nucleic acid molecule as described above can be cloned into any suitable vector and can be used to transform or transfect any suitable host The selection of vectors and methods to construct them are commonly known to persons of ordinary skill in the art and are descnbed m general technical references (see, m general, "Recombinant DNA Part D," Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987) and the references cited herein under "Examples"). Desirably, the vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA or RNA. Preferably, the vector comprises regulatory sequences that are specific to the genus of the host. Most preferably, the vector comprises regulatory sequences that are specific to the species of the host.

Constructs of vectors, which are circular or linear, can be prepared to contain an entire nucleic acid sequence as described above or a portion thereof ligated to a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived from ColEl, 2 mμ plasmid, λ, SV40, bovine papilloma virus, and the like.

In addition to the replication system and the inserted nucleic acid, the construct can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like.

Suitable vectors include those designed for propagation and expansion or for expression or both. A preferred cloning vector is selected from the group consisting of the pUC series the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clonetech, Palo Alto, CA). Bacteriophage vectors, such as λGTIO, λGTl 1, λZapII (Stratagene), λ EMBL4, and λ NM1149, also can be used. Examples of plant expression vectors include pBIlOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clonetech, Palo Alto, CA). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clonetech).

An expression vector can comprise a native or normative promoter operably linked to an isolated or purified nucleic acid molecule as described above. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the skill in the art. Similarly, the combining of a 12 nucleic acid molecule as described above with a promoter is also within the skill in the art.

The present invention not only provides a vector comprising an isolated or purified nucleic acid molecule as described above but also provides a vector comprising or encoding an antisense sequence that hybridizes to or a ribozyme that cleaves an RNA molecule encoding PMF- 1. The present invention also provides the antisense molecules, which preferably are at least about 20 nucleotides in length, and the ribozymes, which preferably comprise at least about 20 continuous nucleotides complementary to the target sequence on each side of the active site of the ribozyme. Also in view of the above, the present invention provides a host cell comprising an isolated or purified nucleic acid molecule or a vector as described above. Examples of host cells include, but are not limited to, a human cell, a human cell line, adenovirus, adeno-associated virus, Rous sarcoma virus, mouse mammary tumor virus, Epstein bar virus, E. coli, B. subtilis, P. aerugenosa, S. cerevisiae, and N. crassa. E. coli, in particular E. coli TB-1, TG-2, DH5α, XL-Blue MRF' (Stratagene), SA2821 and Y1090 are preferred hosts.

The present invention further provides an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding human PMF- 1 or a fragment thereof, either one of which is optionally glycoslyated, amidated, carboxylated, phosphorylated, esterified, Ν-acylated or converted into an acid addition salt.. Preferably, the isolated or purified polypeptide molecule (i) consists essentially of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof comprising at least 8 amino acids or (ii) shares 40% or more identity with SEQ ID NO: 2 or a fragment thereof. Also provided is an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding a variant PMF-1 or a fragment thereof comprising at least 8 amino acids, which is optionally glycoslyated, amidated, carboxylated, phosphorylated, esterified, N-acylated or converted into an acid addition salt.

The polypeptide preferably comprises an amino end and a carboxyl end. The polypeptide can comprise D-amino acids, L-amino acids or a mixture of D- and L-amino acids. The D-form of the amino acids, however, is particularly preferred since a polypeptide compnsed of D-amino acids is expected to have a greater retention of its biological activity in vivo, given that the D-ammo acids are not recognized by naturally occurnng proteases

The polypeptide can be prepared by any of a number of conventional techniques. The polypeptide can be isolated or purified from a naturally occurring source or from a recombinant source For instance, m the case of recombinant polypeptides, a DNA fragment encoding a desired peptide can be subcloned into an appropnate vector using well-known molecular genetic techniques (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spnng Harbor Laboratory, 1989)). The fragment can be transcnbed and the polypeptide subsequently translated in vitro. Commercially available kits can also be employed (e.g., such as manufactured by Clontech, Palo Alto, CA. Amersham Life Sciences, Inc., Arlington Heights, IL, InVitrogen, San Diego, CA, and the like) The polymerase chain reaction optionally can be employed m the manipulation of nucleic acids. In addition, the polypeptide or fragment thereof can be glycosylated m accordance with methods known m the art.

Alterations of the native am o acid sequence to produce vanant polypeptides can be done by a vanety of means known to those skilled m the art. For instance, site-specific mutations can be introduced by hgatmg into an expression vector a synthesized oligonucleotide compnsmg the modified site. Alternately, ohgonucleotide-directed site- specific mutagenesis procedures can be used such as disclosed m Walder et al, Gene, 42, 133 (1986), Bauer et al., Gene, 37, 73 (1985); Craik, Biotechniques. 12-19 (January 1995), and U S Patents 4,518,584 and 4,737,462

With respect to the above isolated or punfied polypeptides, it is preferred that am such insertions, deletions and/or substitutions are introduced into the amino- terminal region of the PMF-1 polypeptide or vanant thereof and that no insertions, deletions and/or substitutions are introduced into the carboxy-termmal region, particularly the coiled-coil region, of the PMF-1 polypeptide or variant thereof

Any appropnate expression vector (e g., as descnbed m Pouwels et al., Cloning Vectors A Laboratory Manual (Elsevior, NY: 1985)) and corresponding suitable host can be employed for production of recombmant polypeptides. Expression hosts mclude, but are not limited to, bactenal species withm the genera Escherichia, Bacillus, Pseudomonas, Salmonella, mammalian or insect host cell systems including baculovirus systems (e.g., as described by Luckow et al., Bio/Technology, 6, 47 (1988)), and established cell lines such as the COS-7, C127, 3T3, CHO, HeLa, BHK cell line, and the like. The ordinary skilled artisan is, of course, aware that the choice of expression host has ramifications for the type of polypeptide produced. For instance the glycosylation of polypeptides produced in yeast or mammalian cells (e.g., COS-7 cells) will differ from that of polypeptides produced in bacterial cells such as Escherichia coli.

Alternately, the polypeptide (including the variant peptides) can be synthesized using standard peptide synthesizing techniques well-known to those of skill in the art (e.g., as summarized in Bodanszky, Principles of Peptide Synthesis, (Springer- Verlag, Heidelberg: 1984)). In particular, the polypeptide can be synthesized using the procedure of solid-phase synthesis (see, e.g., Merrifield, J. Am. Chem. Soc, 85, 2149-54 (1963); Barany et al., Int. J. Peptide Protein Res., 30, 705-739 (1987); and U.S. Patent 5,424,398). If desired, this can be done using an automated peptide synthesizer. Removal of the t-butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups and separation of the polypeptide from the resin can be accomplished by, for example, acid treatment at reduced temperature. The polypeptide-containing mixture can then be extracted, for instance, with dimethyl ether, to remove non-peptidic organic compounds, and the synthesized polypeptide can be extracted from the resin powder (e.g., with about 25% w/v acetic acid). Following the synthesis of the polypeptide, further purification (e.g., using high performance liquid chromatography (HPLC)) optionally can be done in order to eliminate any incomplete polypeptides or free amino acids. Amino acid and/or HPLC analysis can be performed on the synthesized polypeptide to validate its identity. For other applications according to the invention, it may be preferable to produce the polypeptide as part of a larger fusion protein, either by chemical conjugation, or through genetic means, such as are known to those skilled in the art.

If desired, the polypeptides of the invention (including variant polypeptides) can be modified, for instance, by glycosylation, amidation, carboxylation, or phosphorylation, or by the creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives of the polypeptides of the invention. The polypeptides also can be modified to create polypeptide derivatives by forming covalent or noncovalent complexes with other moieties in accordance with methods known in the art. Covalently-bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the polypeptides, or at the N- or C-terminus. Thus, in this regard, the present invention also provides a conjugate comprising an above-described isolated or purified polypeptide molecule or fragment thereof and a targeting moiety. Preferably, the targeting moiety is an antibody or an antigenically reactive fragment thereof. Alternatively, the targeting moiety can be a reporter group, including, but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes a colorimetric or fluorometric reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin). Methods of conjugation are known in the art. In addition, conjugate kits are commercially available.

The present invention also provides a composition comprising a pharmaceutically acceptable carrier and either (i) an above-described isolated or purified nucleic acid molecule or fragment thereof, (ii) an above-described vector, (iii) an above-described polypeptide molecule or fragment thereof, or (iv) an above- described conjugate comprising an above-described isolated or purified polypeptide molecule or fragment thereof and a targeting moiety. Pharmaceutically acceptable carriers are well-known in the art, and are readily available. The choice of carrier will be determined in part by the particular route of administration and whether a nucleic acid molecule or a polypeptide molecule (or conjugate thereof) is being administered. Accordingly, there is a wide variety of suitable formulations for use in the context of the present invention, and the invention expressly provide a pharmaceutical composition that comprises an active agent of the invention and a pharmaceutically acceptable carrier therefor. The following methods and carriers are merely exemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound dissolved in diluent, such as water, saline, or orange juice; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, 16 corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth.

Pastilles can comprise the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients/carriers as are known in the art.

An active agent of the present invention, either alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also can be formulated as pharmaceuticals for non-pressured preparations such as in a nebulizer or an atomizer. Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Additionally, active agents of the present invention can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. Further suitable formulations are found in Remington's Pharmaceutical Sciences, 17th ed., (Mack Publishing Company, Philadelphia, Pa.: 1985), and methods of drug delivery are reviewed in, for example, Langer, Science, 249, 1527-1533 (1990).

Further provided by the present invention is a hybridoma cell line that produces a monoclonal antibody that is specific for an above-described isolated or purified polypeptide molecule. Methods of making hybridomas are known in the art (see, e.g., the references cited herein under "Examples."). Thus, the present invention also provides the monoclonal antibody produced by the hybridoma cell line. Similarly, the present invention provides a polyclonal antiserum raised against an above-described isolated or purified polypeptide molecule. Methods of raising polyclonal antiserum against a polypeptide molecule are also known in the art (see, e.g., the references cited herein under "Examples."). See, also, Example 13 herein.

A method of regulating a PMF- 1 -responsive gene in a cell is also provided by the present invention. The method comprises contacting the cell with a gene- regulating amount of PMF-1, whereupon the PMF-1 -responsive gene in the cell is regulated. Whether or not a gene is PMF-1 responsive can be determined by measuring the level of gene product in the absence and presence of exogenously added PMF-1, whether in the form of a polypeptide (or, e.g., liposomal formulation thereof) or a nucleic acid, wherein an increase or decrease in the level of gene product in the presence of exogenously added PMF-1 indicates that the gene is PMF-1 responsive. Preferably, the PMF-1 responsive gene encodes SSAT. Preferably, PMF-1 regulates the PMF-1 responsive gene by increasing the transcription of the gene.

The cell is contacted with a gene-regulating amount of PMF- 1 by contacting the cell with an above-described isolated or purified nucleic acid molecule (or functional fragment thereof), an above-described isolated or purified polypeptide molecule (or functional fragment thereof) or an above-described conjugate. Herein, "contacting" is intended to mean that the cell, whether an individual cell or a collection of cells in the form of a tissue, organ or organism, is brought into contact with the isolated or purified nucleic acid molecule (or functional fragment thereof) or polypeptide molecule (or functional fragment thereof) in such a manner that the nucleic acid molecule (or functional fragment thereof), such as in the form of a vector, enters the cell and is expressed therein or the polypeptide molecule (or functional fragment thereof) is taken 18 up by the cell. The cell can be contacted with PMF-1 by any suitable manner, including by in vivo, in vitro and ex vivo methods.

By "functional fragment thereof is meant that the fragment has activity characteristic of PMF-1. In other words, it regulates a PMF-1 responsive gene. The PMF- 1 fragment, however, can be more or less active than the complete PMF- 1 as desired in accordance with the present invention. Desirably, the functional fragment comprises at least the 3 ' end, particularly that which encodes the coiled-coil region, of a nucleic acid molecule encoding PMF- 1 or a variant thereof or the carboxy- terminal end of PMF- 1 or a variant thereof. If the gene is one that typically is upregulated by PMF- 1 , the use of an isolated or purified nucleic acid molecule (or functional fragment thereof) or polypeptide molecule (or functional fragment thereof) that increases PMF-1 m the cell will serve to upregulate the PMF- 1 -responsive gene. By contrast, the use of an antisense nucleic acid molecule, a ribozyme, an isolated or purified nucleic acid molecule (or fragment thereof) that encodes a competitive inhibitor of PMF-1 , or a competitively inhibiting vanant PMF-1 polypeptide (or fragment thereof) will serve to downregulate the PMF- 1 -responsive gene.

If the gene is one that typically is downregulated by PMF-1, the use of an isolated or purified nucleic acid molecule (or functional fragment thereof) or polypeptide molecule (or functional fragment thereof) that increases PMF-1 m the cell will serve to downregulate the PMF- 1 -responsive gene. By contrast, the use of an antisense nucleic acid molecule, a ribozyme, an isolated or purified nucleic acid molecule (or fragment thereof) that encodes a competitive inhibitor of PMF-1, or a competitively inhibiting vanant PMF- 1 polypeptide (or fragment thereof) will serve to upregulate the PMF- 1 -responsive gene.

The nucleic acid sequence introduced in antisense suppression generally is substantially identical to at least a portion, preferably at least about 20 contiguous nucleotides, of the PMF-1 gene, but need not be identical. The vectors can, thus be designed such that the inhibitory effect applies to other proteins withm a family of genes exhibiting homology or substantially homology to the target gene. The introduced sequence also need not be full-length relative to either of the pnmary transcnption product or fully processed mRNA Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same mtron or exon pattern, and homology of non-codmg segments will be equally effective. As an alternative to antisense suppression, interfering RNA can be used to achieve the same effect by a different mechanism of action.

Ribozymes can be designed such that they specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out thrs cleavage, the nbozyme is not itself altered and is, thus, capable of recycling and cleaving other molecules, making it a true enzyme The inclusion of ribozyme sequences with antisense RNAs confers RNA-cleavmg activity upon them, thereby increasing the activity of the constructs The design and use of target RNA-specific nbozymes rs descnbed m Haseloff et al, Nature 334: 585-591 (1988). Preferably, the nbozyme compnses at least about 20 contiguous nucleotides complementary to the target sequence on each side of the active site of the nbozyme

The above-described isolated or purified nucleic acid molecule or functional fragment thereof preferably is operably linked to a promoter. Preferably, the isolated or punfied nucleic acid molecule or functional fragment thereof is m the form of a vector Whether or not the nucleic acid molecule or functional fragment thereof is necessarily in the form of a vector depends, m part, on the particular method (e.g., transformation, transduction, electroporation, micromjection; etc.) used to contact the cell as is known in the art The isolated or purified polypeptide molecule or functional fragment thereof can be contained withm a hposome compnsing a cell-surface targeting moiety that binds to the cell being contacted.

Preferably, the cell that is contacted is a cancerous cell and the method results in a decrease in the viability and/or metastatic potential of the cancerous cell. Preferably, when the cell is a cancerous cell, the method further comprises the simultaneous or sequential administration, whether by the same or different routes, of an anti-cancer agent, such as a chemotherapeutic agent, e.g., a polyamine or an analogue thereof Examples of therapeutic polyammes include those set forth m U.S. Patent Nos. 5,880,161, 5,541,230 and 5,962,533, Saab et al, J Med Chem. 36: 2998- 3004 (1993), Bergeron et al., J Med Chem 37(21) 3464-3476 (1994), Casero et al., Cancer Chemother Pharmacol 36: 69-74 (1995), Bernacki et al., Clin. Cancer Res. 1 : 847-857 (1995); Bergeron et al., J. Med Chem. 40. 1475-1494 (1997); Gabnelson et al., Clinical Cancer Res 5. 1638-1641 (1999), Bergeron et al., J. Med. Chem. 43. 224- 235 (2000), and McCloskey et al., "Altered Spermidme/Spermme'-acetyltransferase activity as a mechanism of cellular resistance to bιs(ethyl)polyamme analogues," J Biol Chem (July 7, 2000), which can be administered alone or in combination with other active agents, such as anti-cancer agents, e.g., cis-diammedichloroplatmum (II) and l,3-bιs(2-chloroethyl)-l-nιtrosourea.

Preferred routes of administration m the treatment of cancer include mtratumoral and pentumoral Also preferred is administration that is targeted to a cancer cell In this regard, examples of cancer-specific, cell-surface molecules mclude placental alkaline phosphatase (testicular and ovanan cancer), pan carcinoma (small cell lung cancer), polymorphic epithelial mucm (ovarian cancer), prostate-specific membrane antigen, α-fetoprotem, B-lymphocyte surface antigen (B-cell lymphoma), truncated EGFR (ghomas), ldiotypes (B-cell lymphoma), gp95/gp97 (melanoma), N- CAM (small cell lung carcinoma), cluster w4 (small cell lung carcinoma), cluster 5A (small cell carcinoma), cluster 6 (small cell lung carcinoma), PLAP (semmomas, ovanan cancer, and non-small cell lung cancer), CA-125 (lung and ovanan cancers), ESA (carcinoma), CD 19, 22 or 37 (B-cell lymphoma), 250 kD proteoglycan (melanoma), P55 (breast cancer), TCR-IgH fusion (childhood T-cell leukemia), blood group A antigen m B or O type individual (gastnc and colon tumors), and the like. Examples of cancer-specific, cell-surface receptors include erbB-2, erbB-3, erbB-4, IL-2 (lymphoma and leukemia), IL-4 (lymphoma and leukemia), IL-6

(lymphoma and leukemia), MSH (melanoma), transferrm (ghomas), tumor vasculature integrals, and the like. Preferred cancer-specific, cell-surface receptors include erbB-2 and tumor vasculature mtegnns, such as CD1 la, CD1 lb, CD1 lc, CD18, CD29, CD51, CD61 , CD66d, CD66e, CD106, and CDwl45 There are a number of antibodies to cancer- specific, cell-surface molecules and receptors that are known. C46 Ab (Amersham) and 85A12 Ab (Unipath) to carcmo- embryonic antigen, H17E2 Ab (ICRF) to placental alkaline phosphatase, NR-LU-10 Ab (NeoRx Corp ) to pan carcinoma, HMFC1 Ab (ICRF) to polymorphic epithelial mucm, W14 Ab to B-human choriomc gonadotropm, RFB4 Ab (Royal Free Hospital) to B-lymphocyte surface antigen, A33 Ab (Genex) to human colon carcinoma, TA-99 Ab (Genex) to human melanoma, antibodies to c-erbB2 (JP 7309780, JP 8176200 and JP 7059588), and the like. ScAbs can be developed, based on such antibodies, using techniques known m the art (see for example, Bind et al., Science 242: 423-426 (1988), and Whitlow et al, Methods 2(2)- 97-105 (1991)).

In general, there are a number of databases for gands, binding domains and cell-surface molecules See, for example, ftp://kegg.genome.ad.jp, http //broweb.pasteur.fr/docs/versions, http://ampere.doe- mbι.ucla.edu:8801/dat/dιp.dat or http://bones.biochem.ualberta.ca pedro/rt-l .htmll .

A "gene-regulatmg amount" of PMF-1 is an amount of PMF-1 that alters the expression of the PMF-1 responsive gene. The alteration m expression can be an mcrease m expression or a decrease m expression When the PMF-1 responsive gene is SSAT, desirably expression of the gene is increased, particularly in the presence of a polyamine or an analogue thereof. The dose administered to an animal, particularly a human, m the context of the present invention will vary with the nucleic acid molecule or polypeptide molecule administered, the composition employed, the route of administration, whether individual cells, a tissue, an organ or an organism is bemg contacted, and the particular site being treated.

Generally, when an above-descnbed polypeptide is administered to an animal, such as a mammal, m particular a human, it is preferable that the polypeptide is administered m a dose of from about 1 to about 1,000 rmcrograms of the polypeptide per kg of the body weight of the host per day when given parenterally. However, this dosage range is merely preferred, and higher or lower doses may be chosen m appropnate circumstances. For instance, the actual dose and schedule can vary dependmg on whether the composition is administered in combination with other pharmaceutical compositions, or depending on mtenndividual differences m pharmacokmetics, drug disposition, and metabolism. One skilled m the art easily can make any necessary adjustments m accordance with the necessities of the particular situation. 22 If desired, the half-life of the polypeptide can be increased by conjugation to soluble macromolecules, such as polysaccharides, or synthetic polymers, such as polyethylene glycol, as described, for instance, in U.S. Patents 5,116,964, 5,336,603, and 5,428,130. Alternately, the polypeptides can be "protected" in vesicles composed of substances such as proteins, lipids (for example, liposomes), carbohydrates, or synthetic polymers. If liposomes are employed, liposome delivery can be carried out as described in U.S. Patent 5,468,481, or using liposomes having increased transfer capacity and/or reduced toxicity in vivo (see, e.g., PCT patent application WO 95/21259 and the references cited therein). Furthermore, polypeptides can be administered in conjunction with adenovirus (preferably replication-deficient adenovirus) to allow the intracellular uptake of the polypeptides by adenoviral-mediated uptake of bystander molecules (e.g., as described in PCT patent application WO 95/21259). Similarly, a conjugate as described above or a fusion of a an above-described polypeptide to an antibody (or an antigenically reactive fragment thereof) that recognizes a cell surface antigen; etc. as described below with respect to nucleic acids can be employed to deliver the resultant fusion protein to a specific target cell or tissue (e.g., as described in U.S. Patent 5,314,995).

Those of ordinary skill in the art can easily make a determination of the gene- regulating amount of an above-described isolated and purified nucleic acid molecule to be administered to an animal, such as a mammal, in particular a human. The dosage will depend upon the particular method of administration, including any vector or promoter utilized. For purposes of considering the dose in terms of particle units (pu), also referred to as viral particles, it can be assumed that there are 100 particles/pfu (e.g., lxlO¹² pfu is equivalent to lxlO¹⁴ pu). An amount of recombinant virus, recombinant DNA vector or RNA genome sufficient to achieve a tissue concentration of about 10² to about 10¹² particles per ml is preferred, especially of about 10⁶ to about 10¹⁰ particles per ml. In certain applications, multiple daily doses are preferred. Moreover, the number of doses will vary depending on the means of delivery and the particular recombinant virus, recombinant DNA vector or RNA genome administered. A targeting moiety also can be used in the contact of a cell with an above- described isolated or purified nucleic acid molecule. In this regard, any molecule that 23 can be linked with the therapeutic nucleic acid directly or indirectly, such as through a suitable delivery vehicle, such that the targeting moiety binds to a cell-surface receptor, can be used The targeting moiety can bind to a cell through a receptor, a substrate, an antigenic determinant or another binding site on the surface of the cell. Examples of a targeting moiety include an antibody (i.e , a polyclonal or a monoclonal antibody), an lmmunologically reactive fragment of an antibody, an engineered lmmunoprotem and the like, a protein (target is receptor, as substrate, or regulatory site on DNA or RNA), a polypeptide (target is receptor), a peptide (target is receptor), a nucleic acid, which is DNA or RNA (i.e., smgle-stranded or double- stranded, synthetic or isolated and punfied from nature; target is complementary nucleic acid), a steroid (target is steroid receptor), and the like In general, there are a number of databases for targeting moieties (see, e.g , ftp://kegg.genome.ad.jp, http //broweb pasteur fr/docs/versions, httpV/ampere.doe- mbι.ucla.edu:8801/dat/dιp.dat, or http://bones.biochem.ualberta.ca pedro/rt-l .htmll). Analogs of targeting moieties that retain the ability to bind to a defined target also can be used. In addition, synthetic targeting moieties can be designed, such as to fit a particular epitope. Alternatively, the therapeutic nucleic acid can be encapsulated in a hposome comprising on its surface the targeting moiety

The targeting moiety includes any linking group that can be used to join a targeting moiety to, m the context of the present invention, an above-descnbed nucleic acid molecule. It will be evident to one skilled in the art that a vanety of linking groups, including bifunctional reagents, can be used. The targeting moiety can be linked to the therapeutic nucleic acid by covalent or non-covalent bonding. If bonding is non-covalent, the conjugation can be through hydrogen bonding, ionic bonding, hydrophobic or van der Waals interactions, or any other appropnate type of binding. A method of assessing the sensitivity of cells to treatment with a polyamine or an analogue thereof is also provided by the present invention The method compnses assessing the level of PMF-1 RNA or PMF-1 polypeptide m a sample of the cells after contact with a polyamine or an analogue thereof. Methods of assessing mRNA and polypeptide levels are known m the art and are descnbed m the references cited herein under "Examples " An increase in the level of PMF-1 mRNA or PMF-1 24 polypeptide in the sample of cells after contact with a polyamine or an analogue thereof as compared to a control sample or a sample of the cells before contact with the polyamine or analogue thereof is indicative of sensitivity of the cells to treatment with a polyamine or an analogue thereof. Preferably, the cells are cancerous. Thus, a method of assessing the effectiveness of treatment of cells with a polyamine or an analogue thereof is also provided. The method comprises assessing the level of PMF-1 mRNA or PMF-1 polypeptide in a sample of the cells before and during treatment of the cells with a polyamine or an analogue thereof. Methods of assessing mRNA and polypeptide levels are known in the art and are described in the references cited herein under "Examples." An increase in the level of PMF-1 mRNA or PMF-1 polypeptide in the cells during treatment of the cells with a polyamine or an analogue thereof is indicative of efficacy of treatment of the cells with a polyamine or an analogue thereof.

See U.S. Patent No. 5,498,522 for methods for the use of SSAT as a prognostic indicator and/or tissue response marker, which methods can be adapted for use with PMF-1.

25 EXAMPLES The following examples serve to illustrate the present invention and are not intended to limit its scope in any way.

N¹,Nⁿ-Bis(ethyl)norspermine (BENSpm) was kindly provided by Parke-Davis. 2-Difluoromethylornithine was obtained as a gift from the Marion-Merrell-Dow Research Institute (Cincinnati, OH). Radionucleotides, [α-³²P]dCTP, and [α- ^3:>S]methionine were supplied by Amersham Pharmacia Biotech (Arlington Heights, IL). A human placental retro viral cDNA library, the Matchmaker® yeast two-hybrid system, yeast culture media, and a human multiple-tissue Northern blot system were purchased from Clontech (Palo Alto, CA). QuikChange® Site-Directed Mutagenesis Kit was purchased from Stratagene (LaJolla, CA). A human bacterial artificial chromosomal (BAC) DNA library was obtained from Research Genetics (Huntsville, AL). Lipofectin reagent was purchased from Life Technologies, Inc. (Rockville, MD). The luciferase assay system, TnT coupled transcription/translation reticulocyte lysate systems, and the cDNA synthesis system were purchased from Promega (Madison, WI), and the Gal-XE chemiluminescent reporter gene assay system was purchased from ICN Pharmaceuticals (Cosa Mesa, CA). Restriction and DNA-modifying enzymes were purchased from Life Technologies, Inc., New England Biolabs Inc. (Beverly, MA), and Sigma (St. Louis, MO). Oligo(dT)-cellulose was purchased from Roche Molecular Biochemicals (Indianapolis, IN). The TA cloning kit was purchased from Invitrogen (Carlsbad, CA). All oligonucleotides used in the experiments were synthesized by Life Technologies, Inc. Other chemicals were purchased from Sigma, Roche Molecular Biochemicals, and J. T. Baker, Inc. (St. Louis, MO).

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference:

Birren et al., Genome Analysis: A Laboratory Manual Series, Volume 1, Analyzing DNA, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1997), 26 Birren et al., Genome Analysis: A Laboratory Manual Series, Volume 2, Detecting Genes, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998),

Birren et al., Genome Analysis: A Laboratory Manual Series, Volume 3, Cloning Systems, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999),

Birren et al., Genome Analysis: A Laboratory Manual Series, Volume 4, Mapping Genomes, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999), Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY (1988),

Harlow et al., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999),

Hoffman, Cancer and the Search for Selective Biochemical Inhibitors, CRC Press (1999),

Pratt, The Anticancer Drugs, 2nd edition, Oxford University Press, NY (1994), QIAexpress Detection and Assay Handbook, 2nd edition, QIAGEN Inc., 28159 Avenue Stanford, Valencia, CA 91355 (April 1999),

(http://www.qiagen.com/literature/handbooks/qxp/qxpda/qxpda.pdf), and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold

Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

EXAMPLE 1

This example describes the identification of PMF-1 as a co-transcription partner ofNrf-2.

A 2017-bp Nrf-2 cDNA (+27 to +2043 bp) fragment was derived from mRNA from the human non-small cell lung cancer cell line H157 (National Cancer Institute, Bethesda, MD) using reverse-transcription polymerase chain reaction (PCR). The PCR product was then ligated into the vector pCR2.1 to generate the plasmid pCR2. l/Nrf-2. The Nrf-2 cDNA fragment was then cut from pCR2. l/Nrf-2 using Kpn 27 I and Eco RV and was inserted into the vector pcDNA3.1(+), which had been digested with the same restriction enzymes, to construct the plasmid pcDN A3. l/Nrf-2.

The plasmid pcDNA3.1 /PMF-1 was constructed by cutting the PMF-1 cDNA from pCR2.1/PMF- 1 with Eco RI and Xho I and cloning the PMF- 1 cDNA into the pcDNA3.1 (+) vector, which had been digested with the same restriction enzymes. The pBKH-93 plasmid was constructed as previously reported (Wang et al. (1998), supra). pBKH-93/dPRE was constructed by annealing complementary oligonucleotides and filling in the protruding ends with Taq DNA polymerase. The resulting 30-bp double-stranded DNA fragment containing two PRE elements in the same direction was cloned into the vector pCR2.1. The PRE-containing fragment was then excised by using Kpn I and Xho I and inserted into the same restriction enzyme sites in pBKH-93.

Yeast two-hybrid screening was performed with the Matchmaker two-hybrid system. Bait plasmids were constructed by producing the coding region of the Nrf-2 cDNA (+39 to +2043 bp) and the leucine zipper domain (+ 1338 to +2043 bp) of the Nrf-2 protein by PCR, using pCR2. l/Nrf-2 as the template. The primers used to amplify full-length Nrf-2 or the Nrf-2 leucine zipper domain were designed to contain anXba I or a Sal I restriction site in the 5'-end, respectively. The PCR products were digested with Xba I and Sal I and then subcloned in-frame into the same restriction enzyme sites in the DNA-binding domain of Gal4 in the vector pAS2.1. In initial experiments, two pAS2.1 constructs were used as bait, i.e., full-length Nrf-2 (amino acids 1-589) and the leucine zipper domain of Nrf-2 (amino acids 434-589). Each construct was fused to the Gal4 DNA-binding domain. However, in testing, the full- length Nrf-2 fusion plasmid (pAS2. l/Nrf-2) strongly activated the lacZ and HIS3 reporter genes in the host Y 190 cells in the absence of the Gal4 transcriptional activation domain. Consequently, the bait plasmid containing only the leucine zipper domain of Nrf-2 (pAS2.1/Nrf-2-LZ) was used for further experimentation.

The pACT-2/H157 cDNA library was constructed by synthesizing HI 57 cDNA using the Promega cDNA synthesis system according to the manufacturer's protocol. The double-stranded cDNA was ligated to Eco RI adapters on both ends and then cloned into the Gal4 transcriptional activation domain in the pACT-2 vector, which had 28 been digested with Eco RI and dephosphorylated with calf intestine alkaline phosphatase. The pACT-2/Hl 57 cDNA library was then screened by the "HIS3 Jump- Start" procedure according to the protocol from the manufacturer.

Saccharomyces cerevisiae Y190 cells were first transformed with bait plasmids and selected on synthetic dextrose medium lacking tryptophan (SD— Trp). The transformants grown on the SD— Trp medium were subsequently transformed with the pACT-2/H157 cDNA library and selected on medium lacking tryptophan and leucine (SD— Trp-Leu). The clones co-transformed with the bait and library were collected and replated onto medium lacking tryptophan, leucine, and histidine (SD~Trp-Leu-His) with 30 mM 3-amino-l ,2,4-triazole to inhibitthe leaking growth of Y190 cells. The clones selected in this step were further assayed for their β-galactosidase activity. Thirty three clones from approximately 10⁶ yeast transformants were identified that could activate the reporter genes. The pACT-2 library plasmids were purified from individual positive clones and amplified in Escherichia coli. Inserts of the positive clones were amplified by PCR with Matchmaker 5" and

3" BD insert screening amplimers and digested with the restriction enzyme Alu I. Gel electrophoresis/ethidium bromide staining results demonstrated an identical insert of approximately 1 kb, which was designated as PMF-1.

In order to confirm the interaction between the Nrf-2 leucine zipper domain and PMF-1, Y190 yeast cells were transformed with pAS2.1/Nrf-2-LZ alone, pACT- 2/PMF-l alone, or pAS2.1/Nrf-2-LZ and pACT-2/PMF-l together. Transformants were then selected on SD-His-Leu-Trp medium containing 30 mM 3 -amino- 1,2,4- triazole. Only clones co-transformed with pAS2.1/Nrf-2-LZ and pACT-2/PMF-l grew on the selection medium, demonstrating transcriptional activation of the reporter gene. The cDNA insert in the positive clone pACT-2/PMF-l was sequenced with a

Perkin-Elmer ABI Automated DNA Sequencer. Sequencing of the cDNA insert revealed a 958 bp cDNA that encoded a novel Nrf-2 partner, which was designated PMF-1.

Example 2

This example describes the cloning of human PMF-1 cDNA. 29 A primer representing the 5'-pLIB vector sequence upstream from the multiple cloning site, and another primer corresponding to sequence in the 3'-end of PMF-1 cDNA (997-1020 bp) were used to clone the PMF-1 cDNA from a human placental retroviral cDNA library into the pLIB vector (Clontech) by PCR. This PCR product was then subcloned into the pCR2.1 vector (pCR2.1 /PMF-1). Using this method, a transcript of 1057 bp was identified (SEQ ID NO: 1). This clone contained 99 more bases in the 5' end as compared with the cDNA obained from the yeast two-hybrid assay. The message size correlated well with the mRNA length estimated by Northern blotting (see Example 6). PMF-1 cDNA contains an open reading frame of 495 bp and is predicted to encode for a protein of 165 amino acids (SEQ ID NO: 2) with a calculated molecular mass of 19.2 kDa. The sequence surrounding the translational initiation codon is a nearly perfect consensus with the Kozak translational consensus sequence, with 9 out of 10 matches for the proposed optimal context. The polyadenylation signal sequence is found at nucleotide 1025, 13 bp upstream from the poly(A) tail. A leucine zipperlike structure was found in the N-terminal region.

Example 3

This example describes the cloning of human genomic PMF-1. A human BAC library was screened to obtain the PMF-1 genomic sequence using the PCR protocol supplied by the manufacturer. Three pairs of primers used in the PCR screening were designed according to PMF-1 cDNA sequence.

Screening at three consecutive levels of the library (BAC super pool library, BAC plate pool library, and BAC single clone library) resulted in the discovery of a positive clone, P-6, which was used as a template for all of the three primer pairs. In order to facilitate plasmid preparation and sequencing, the DNA insert in the P-6 clone was digested with Hin dill and subcloned into the pBluescript SK vector. The P-6 subclone library was then screened for the clones containing PMF-1 genomic fragments (clones A, B and E) by colony lift assay and Southern blot analysis with random primer-labeled PMF-1 cDNA probe. Clones A, B, and E were found to contain the PMF-1 genomic fragments. The identification of DNA sequences of clones 30 technique with the P-6 plasmid as the template.

The DNA inserts in the positive clones were sequenced on the ABI Automated DNA Sequencer. The overall length of the assembled human PMF-1 genomic sequence (SEQ ID NO: 3) from the above clones is -28 kb. These clones span the entire cDNA region of PMF-1 and define the five complete exons (see Tables I and II) and fourintrons. Clone A also defines 11.2 kb upstream from the transcriptional start site. All of the splice junctions in the PMF-1 gene conform to consensus sequence established for splice donor and splice acceptor sites. Exon 1 contains 111 bp of 5'- untranslated sequence and the first 41 bp of coding sequence. Exon 5 contains the 0 sequence coding for the last 17 amino acids of PMF-1, the stop codon (TGA), and a 434-bp 3'-untranslated sequence of the PMF-1 cDNA.

Table I.

5 Exon-Intron Organization of the Hnman PMF-1 Gene

Exon Exon Sequence at ezon-intron junction* Intron Codons no. ιize,bp 5¹- splice donor 3^* splice acceptor rize,bp interrupted

1 152 GGC AG gtaaag-- — -cgcscag C TAC -20 Kb Ser" 2 106 AZC CGG gtgagt— — cttcatj GAG GAA 1203 Arg"-Glιr so 3 101 βCC TG gtgaga- — -ctccag G CGC 2559 Trp^M 0 196 TGG CAG gtcagt— — -tttcag GCT CXλ 3066 Gln^-Ala¹"

* Exon sequences are in uppercase letters and intron sequences are in lowercase letters.

Table II. 5

Exon Borders for AH008078 (Human PMF-1 gene)

Exon l . 11217 to 11368

Exon 2. 30604 to 30709

Exon 3. 31913 to 32013

Exon 4. 34573 to 34768

Exon 5. 37835 to 38322 31 The TESS program (URL: http://dot.imgen.bcm.tm-c.edu:9331/ under "Gene Features Search") was used to identify putative binding sites for known transcription factors in the PMF-1 promoter region 2 kb immediately upstream from the transcriptional start site. It should be noted that one potential PRE-binding site (Wang et al. (1998), supra) was identified in this region (-1450 to -1442 bp). This site has 8 out of 9 base pairs identical to the previously described PRE (Wang et al. (1998), supra) and oriented in the antisense direction. Although several putative transcription factor-binding sites have been identified, further experimentation will be necessary to determine whether any of the identified sites have any actual function. It should be noted that, similar to SSAT (Xiao et al., Biochem. Biophys. Res. Commun. 187: 1493- 1502 (1992)), the transcription of PMF-1 is driven by a TAT A-less promoter.

Example 4

This example describes the chromosomal location of human PMF-1. The radiation hybrid screening technique with the Stanford G3 RH panel

(Research Genetics) was used to determine the chromosomal location of PMF-1 as per the supplier's PCR protocol. Primers spanning from within exon 4 into intron 4 were used. Analysis of results was performed using the protocol at the Stanford web site (http://shgc.stanford.edu). The results indicate that the PMF-1 gene is on the long arm of chromosome 1 at the lql2 locus. Since this region represents the large pericentric heterochromatin of lq and the resolution of the markers determined from the radiation hybrid screening was not sufficient to precisely place PMF- 1 , fluorescent in situ hybridization analysis was performed as previously reported (Xiao et al. (1992), supra) using the labeled 5-kb B and 10-kb E fragments as probes to confirm the location of PMF-1. The results of 22 G-banded metaphase chromosome pairs indicated PMF-1 to be at the Iql2/lq21 border (14 of 22 signals were at the Iql2/lq21 border). Example 5

This example describes in vitro transcription and translation of PMF-1 cDNA.

In vitro transcription and translation were performed with the TnT coupled transcription/translation lysate system using [α-³⁵S]methionine according to the manufacturer's protocol. Purified plasmid pcDN A3.1 /PMF-1 was used as the template. The labeled translation products were separated by 15% SDS-polyacrylamide gel electrophoresis and exposed to Kodak X-Omat film. In vitro transcription and translation of full-length PMF-1 cDNA produced a major band with an apparent molecular mass of -20 kDa, which agrees with the predicted open reading frame.

Example 6

This example describes the expression of PMF-1 mRNA in a variety of normal human tissues.

Total cellular RNA from HI 57 and H82 (a human small cell lung cancer cell line available from the National Cancer Institute, Bethesda, MD) cells was extracted using the acid phenol-guanidine isothiocyanate method (Chomczynski et al., Anal. Biochem.162: 156-159 (1987)). Poly(A⁺) RNA was isolated using oligo(dT)-cellulose chromatography following the manufacturer's directions.

Ten μg of total RNA from HI 57 or H82 cells were separated on a denaturing 1.5% agarose gel containing 6% formaldehyde, transferred to GeneScreen membrane (NEN Life Science Products, Boston, MA), and hybridized with a random primer- labeled PMF-1 cDNA as a probe. Blots were washed and reprobed with a 28 S ribosomal cDNA probe as a loading control. The human multiple-tissue Northern blot system was used to examine the expression of PMF-1 in various human tissues according to the manufacturer's protocol. A 1.2-kb mRNA transcript of PMF- 1 was observed and appears to be expressed almost ubiquitously, although at different levels, in multiple tissues. The tissues expressing the highest levels of PMF-1 were tissues that are highly differentiated and generally not proliferating. The heart and skeletal muscle were among the highest in PMF-1 expression, with significant levels expressed in the kidney and liver. These results suggest that high expression of PMF-1 is associated with low proliferative activity. The exception to this, of course, is the liver, which can proliferate if injured. Example 7

This example describes the expression of PMF-1 mRNA m response to polyamine analogue exposure m human lung cancer cells.

In order to determine if PMF- 1 expression can be induced by treatment with the polyamine analogue BENSpm in a cell type-specific manner, total RNA from the analogue-sensitive HI 57 and analogue-insensitive H82 cells was analyzed by Northern blot analysis after a 24-h exposure to 10 μM BENSpm Significant induction of PMF- 1 mRNA was detected only m the polyamine analogue-sensitive cell line, HI 57 A time-course analysis of BENSpm exposure was performed in order to characterize further the expression of PMF-1 in HI 57 cells PMF-1 was induced m a biphasic manner, peaking first at ~4 h post- treatment and reaching levels >5-fold after a 24-h exposure to BENSpm.

Example 8 This example demonstrates that PMF-1 can induce PRE-mediated transcnption of the SSAT promoter.

In order to determine whether or not PMF-1 could induce PRE-mediated transcription of the SSAT promoter, two reporter plasmids were constructed. One plasmid, pBKH-93, contained the minimal promoter region of the SSAT gene (-93 to -1 bp) upstream from the luciferase gene The other plasmid, pBKH-93/dPRE, contained the minimal promoter region and a 30-bp oligonucleotide containing two PRE consensus sequences (Wang et al (1998), supra) cloned into a site upstream from the minimal SSAT promoter in pBKH-93 For expression of Nrf-2 and PMF- 1 , the cDNAs of Nrf-2 and PMF-1 were cloned into the pcDNA3.1 vector. For transient transfection, 2 * 10⁵ HI 57 cells were seeded m a 35-mm diameter culture dish and cultured in RPMI 1640 medium containing 5 mM 2- difluoromethylormthme for 48 hr to reduce endogenous polyammes and background transcription, as previously reported (Wang et al. (1998), supra). Lipofectm-mediated transfection was performed with 1.5 μg of luciferase reporter constructs and 0.4 μg of control plasmid pSV-β-galactosidase, according to the manufacturer's protocol. After a 5-hr incubation, the DNA-Lipofectm complex-containing medium was replaced by RPMI 1640 medium containing 5 mM 2-dιfluoromethylormthme Forty-eight hr after transfection, the cells were exposed to 10 μM BENSpm for 2 hr The cells were harvested, quick- frozen, and subsequently prepared for luciferase activity measurements as per the instructions of the manufacturer In order to account for variations m transfection efficiency, the luciferase activity was normalized to the β- galactosidase activity

A PRE-mediated induction of luciferase expression in the BENSpm-treated HI 57 cell line was demonstrated in the transient transfection assay using the PRE- contammg construct pBKH-93/dPRE Transfection of the reporter construct together with pcDNA3.1 /PMF- 1 (no addition of BENSpm) produced an increase m transcription that was comparable to the increase m luciferase activity observed after the addition of BENSpm without co-transfection with pcDNA3.1 /PMF- 1 Additional expression of Nrf-2 in the presence or absence of PMF-1 had little effect on the reporter construct expression. These results demonstrate that PMF-1, not Nrf-2, is the limiting factor in this system and that an increase m PMF- 1 in the presence or absence of the analogue can lead to a PRE-mediated increase m transcnption

Example 9

This example demonstrates that expression of Nrf-2 alone does not influence the growth rate of H82 cells

H82 cells (2 x 10⁶) were transfected with 2 μg of pcDNA3. l/Nrf-2 or pcDNA3 1 vector by Lipofectin® Reagent (Lifetech). After 5 hr of incubation, the DNA-Lipofectin complex-containing medium was replaced with RPMI 1640 medium containing 10% calf serum Forty eight hours after transfection, the cells were selected with 400 μg/ml G418 Starting with the cells selected with G418 for 15 days, the rate of cell growth was analyzed The results indicated that stable expression of Nrf-2 alone does not influence the growth rate of H82 cells

Example 10 This example demonstrates that expression of PMF-1 alone does not influence the growth rate of H82 cells 35 H82 cells (2 x 10⁶) were transfected with 2 μg of PCEP4/PMF-1 or pCEP5 vector by Lipofectin® Reagent. After 5 hr of incubation, the DNA-Lipofectin complex-containing medium was replaced with RPMI 1640 medium containing 10% calf serum. Forty eight hours after transfection, the cells were selected with 200 μg/ml hygromycin. Starting with the cells selected with hygromycin for 15 days, the rate of cell growth was analyzed. The results indicated that stable expression of PMF-1 alone does not influence the growth rate of H82 cells.

Example 11 This example demonstrates that expression of Nrf-2 and PMF-1 is toxic to H82 cells.

H82 cells (2 x 10⁶) were transfected with (i) 1 μg of pCEP5/PMF-l and 1 μg of pcDNA3. l/Nrf-2 and (ii) 1 μg of pCEP5 and 1 μg of pcDNA3.1. After 5 hr of incubation, the DNA-Lipofectin complex-containing medium was replaced with RPMI 1640 medium containing 10% calf serum. Forty eight hours after transfection, the cells were selected with 200 μg/ml hygromycin and 400 μg/ml G418. The results indicated that, after 25 days of selection with hygromycin and G418, H82 cells co- transfected with two vectors grew but almost all H82 cells co-transfected with Nrf-2 and PMF-1 died, indicating that co-expression of Nrf-2 and PMF-1 is toxic to the H82 cells.

H82 cells were transfected with pCEP4/PMF-l or pCEP4 followed by selection with 200 μg/ml hygromycin for 20 days, H82 cells already transfected with pCEP4/PMF-l were transfected with 2 μg of pcDN A3. l/Nrf-2, and H82 cells already transfected with pCRP4 were transfected with 2 μg of pcDNA3.1. After 5 hr of incubation, DNA-Lipofectin complex-containing medium was replaced with RPMI 1640 medium containing 200 μg/ml hygromycin. Forty eight hours after transfection, the cells were selected with 400 μg/ml G418 and 200 mg/ml hygromycin. Starting from this time point, the rate of cell growth was analyzed. The results indicated that, although some cells survived from the selection with G418 and hygromycin, co- transfection with PMF-1 and Nrf-2 greatly inhibited the growth of H82 cells within 20 36 days of selection, indicating the toxicity of co-expression of Nrf-2 and PMF-1 to H82 cells

Using H82 cells after 20 days of selection with G418 and hygromycin as a starting point, a new cell growth analysis was conducted This time, no difference of growth rate between H82 cells co-transfected with PMF-1 and Nrf-2 and H82 cells co- transfected with pcDNA3.1 and pCEP4 was observed. It is possible that, after 20 days of treatment with G418 and hygromycin, those cells that did not express high levels of PMF-1 and Nrf-2 or those cells that were resistant to the high levels of PMF-1 and Nrf-2 were selected.

Example 12

This example demonstrates that cells transformed with a PMF-1 -expressing vector express PMF-1 at a high level and that the molecular weight of the expressed PMF-1 agrees with the molecular weight predicted from its deduced ammo acid sequence

PQE-30 (Qiagen, Valencia, CA) is a high-level bacterial expression vector m which the expression of a foreign protein is under the control of a phage T5 promoter that is recognized by E. coli RNA polymerase and a synthetic ribosome binding site (RBS II). The expression of the foreign protein is controlled through a double lac operator system withm the promoter region that effectively blocks protein synthesis m the presence of high levels of lac repressor The expression of the foreign protein is induced by the addition of IPTG. Expression levels up to 50% of total cellular protein can be achieved.

M15[pREP4] is an E. coli expression host transformed with pREP4 plasmid, which constitutively expresses the lac repressor at high levels. This strain can stably propagate an expression construct encoding a toxic or hydrophobic protein.

PMF-1 cDNA containing the entire open reading frame was cloned mto pQE30 at the restriction sites Sac I and Hin dill. The construct generates a fusion protein m which 16 more ammo acid residues, including a tag of six contiguous His residues, were added to the N-termmus of PMF-1 (see lAexpress^R Expression System, 37 QIAexpress Type IV Kit, and QlAexpress ^R — The Complete System Ni-NTA Technology and the 6xHιs Tag, which are available from Qiagen)

PQE30/PMF-1 was transformed into M15[pREP4] cells The expression of PMF-1 protein was induced by IPTG (0.2 mM) in the LB medium The expression levels of PMF-1 were determined by SDS-PAGE at 0, 1 , 2, 3, 4 and 5 hrs after addition of IPTG

The results indicate that M15[pREP4] transformed with pQE30/PMF-l can express PMF-1 protein at high levels in the presence of IPTG The expressed PMF-1 has a molecular weight of 21 5 kd, which agrees well with the molecular weight predicted based on the deduced ammo acid sequence

Example 13

This example describes the preparation of antι-PMF-1 antibodies.

Using pGEM-PMF-1 as a template, a PCR reaction was performed to replace the initial ATG codon with an m-frame Bam HI srte. This insertion replaced the initial methionme of PMF-1 with a glycme but left the second senne mtact. The PCR product was then ligated into the pQE-30 vector (Qiagen), which contains a 6xHιs tag at the ammo terminus of the protein coding region The pHIS-PMF-1 C360A mutant was generated by using the chameleon mutagenesis kit (Stratagene) to mutate cysteme 360 to alanme in pGEM-PMF-1 The pGEM construct was then digested with Sph I and Sal I and the fragment containing the mutation was isolated and inserted into pHIS-PMF-1 digested with the same enzymes. Both pHIS-PMF-1 plasmids were transformed into XL 1 -Blue E coli (Stratagene) by electroporation. Cells were grown overnight m a 10 ml culture of LB broth supplemented with 50 μg/ml ampicillin for protein punfication. The saturated 10 ml culture was then added to 490 ml of pre- warmed LB+amp and incubated with shaking at 37°C until reaching a density corresponding to an OD600 of >0.5 Protein expression was induced by the addition of 300 μM IPTG and the cells were allowed to grow for 4 additional hours Cells were harvested by pelleting at 4,000 x g for 10 mm at 4°C. The cell pellet was resuspended in 20 ml of buffer A (20 mM Tns-HCl, pH 8.0; 400 mM NaCl; 5 mM imidazole) and sonicated m four 5 ml aliquots for a total of 3 mm each (10 sec on, 10 sec off) The 38 sonicated cells were then centrifuged for 1 hr at 15,000 x g The supernatant was diluted to 40 ml with buffer A and loaded onto a 1 ml Talon metal chelate column (Invitrogen) The column was washed with 10 bed volumes of buffer A, followed by 20 bed volumes of buffer B (20 mM Tns-HCl, pH 8.0, 400 mM NaCl, 10 mM imidazole) The protein was eluted into 1 ml fractions with buffer C (20 mM Tns- HCl, pH 8 0, 400 mM NaCl, 200 mM imidazole) The fractions were immediately supplemented with DTT to a final concentration of 2.5 mM to stabilize the PMF-1 activity Ahquots of each fraction were run on a 10% SDS-PAGE gel and fractions containing purified PMF-1 (usually fractions 2, 3 and 4) were combined The imidazole was removed using a PD- 10 gel filtration column (Pharmacra) pre- eqmlibrated with PMF-1 assay buffer (25 mM Tns-HCl, pH 7 5, 2.5 mM DTT; 0 1 mM EDTA) The protein was further concentrated, if necessary, using a C-10 centπcon microconcentrator (Amicon) A typical yield of HIS-PMF-1 from 500 ml of culture is approximately 4 mg When assayed under standard conditions, the His- tagged protein has activity comparable to wild-type PMF- 1

Female New Zealand White rabbits were immunized by mtradermal injection at multiple sites with 200 μg ahquots of the protein antigen emulsified with complete Freunds adjuvant Three weeks later, a second immunization with the same amount of antigen m Freunds incomplete adjuvant was given. Two weeks later, a small sample of blood was taken to check that there was a response and the titer was increased by a third immunization with 100 μg of antigen in Freunds incomplete adjuvant Blood was taken 8 days later and at further intervals of about 6 weeks These later bleedings were taken 8 days after re-immunization with the antigen as above in order to boost the antibody titer In order to remove the amme-contammg Tns buffer, purified 6xHιs-PMF-l (4 mg) was first passed through a G25 sepharose column (PD-10, Pharmacia), which had been equilibrated with 0.1 M phosphate buffer, pH 7.5. The PMF-1 protein was then linked to an activated agarose support (Ammolmk column, Pierce) according to the following procedure The column was equilibrated with coupling buffer (0.1 M phosphate, pH 7.0; 0.05% sodium azide) and 2 ml of the PMF-1 protein was added A reducing solution consisting of 64 mg/ml of sodium cyanoborohydnde (0.2 ml) was added to the top of the column and the gel was completely resuspended by inverting the column The column was mixed by end-to- end shaking for two hrs at room temperature, and then washed with coupling buffer. Coupling efficiency was determined by collecting the effluent and assaying for protein using the method of Bradford No detectable protein was found m the effluent, indicating virtually 100% coupling efficiency. In order to block sites not coupled to protein, the gel was incubated for 30 mm at room temperature with quenching buffer (1.0 M Tris-HCl, pH 7 4) containing the same concentration of reducing solution. The column was then washed with 20 ml of wash solution (1.0 M NaCl) and stored m water containing 0.05% sodium azide at 4°C until use.

The PMF-1 affinity column described above was first equilibrated with 6 ml of phosphate -buffered salme (PBS). Rabbit serum containing polyclonal PMF-1 antibodies (1 ml) was applied to the top of the column, followed by 1 ml of PBS, and the column was incubated for 1 hr at room temperature. The column was then washed with 10 column volumes of PBS, and the bound antibody was eluted into 1 ml fractions with 0.1 M glycme, pH 2.8. Fractions were neutralized by the addition of 50 μl of 1M Tns-HCl, pH 9.5, and the protein concentration of each fraction was measured. The antibody was found to elute in fractions 2-4, with a total yield of approximately 150 μg/ml of serum. The column was regenerated by washing with 10 column volumes of PBS, and stored in 0.05% sodium azide The PMF-1 antibody was used for subsequent Western blots at a concentration of 0.5 μg/ml.

Example 14

This example demonstrates that a non-leucme zipper motif that is present m a coiled-coil region of the carboxy terminus of PMF-1 is required for PMF- l/Nrf-2 binding pAS2.1/Nrf-2-LZ from Example 1 was used as the wild-type bait plasmid in the Matchmaker® yeast two-hybrid system. pACT-2/PMF-l from Example 1 was digested with Bam HI and the small DNA fragment that contained 277 bp of the 5' end of PMF-1 cDNA was recovered, cloned into the pACT2 vector digested with the same enzyme and dephosphorylated using calf intestinal alkaline phosphatase. 40 pACT2/PMF-lC was constructed by recovenng the large DNA fragment from the Bam HI digestion of pACT-2/PMF-l (637 bp of the 3' end of PMF-1 cDNA) and circularizing the large DNA fragment with the open pACT2 vector sequence with T4 ligase The mutation plasmids, pACT2/PMF- lMu, pACT2/PMF- 1 CMul ,

_PACT2/PMF-lCMu2, pAS2 l/Nrf-2-LZMul, pAS2 l/Nrf-2-LZMu3 and pAS2 1/Nrf- 2-LZMu4 were constructed using site-directed mutagenesis with the QuikChange® Site-Directed Mutagenesis Kit from Stratagene Briefly, two synthetic oligonucleotide primers containing desired mutations were used m the PCR reaction with the plasmids containing wild-type target cDNA as the templates Following temperature cycling using Pfu Turbo DNA polymerase, the products were treated with Dpn I endonuclease to digest the parental DNA and to select for mutation-contammg DNAs The DNA was recovered and used to transform XL- 12 Blue super-competent E coli for amplification All mutations were verified by DNA sequencing using a Perkm-Elmer ABI automated DNA sequencer

Yeast two-hybnd analysis was performed with the Matchmaker® yeast two- hybnd system m a "HIS3 jump-start" procedure Saccharomyces cerevisiae Y190 cells were first transformed with the bait plasmid (pAS2 l/Nrf-2-LZ, pAS2 l/Nrf-2-LZMul, pAS2.1/Nrf-2-LZMu3 or pAS2.1/Nrf-2-LZMu-4) and selected on synthetic dextrose medium lacking tryptophan (SD-trp) The transformants selected by the SD-Tφ medium were subsequently transformed with the prey plasmid (pACT2/PMF-l , pACT2/PMF-lC, pACT2/PMF-lN, pACT2/PMF-lMu (He 48→Ser and He 55→Ser), pACT2/PMF- lCMul (Gin 113→ Pro and Gin 120→Pro) or pACT2/PMF- 1 CMu2 (Leu 138→Pro and Gin 144— Pro) and selected by the medium lacking tryptophan and leucine (SD-Trp- Leu) The clones co-transformed with the bait and prey were used for the LacZ reporter gene assay or re-plated onto the medium lacking tryptophan, leucine and histidine (SD- Tφ-Leu-His) with 30 mM 3-ammo-l,2,4-tnazole for the HIS3 reporter gene assay

Co-transformation of yeast with pAS2 l/Nrf-2-LZ and pACT 2/PMF-lMu (not self-activating) followed by selection and reporter activity analysis allowed the yeast to grow in selection medium and to activate both of the LacZ and HIS3 reporter genes as 41 effectively as the wild-type construct Thus, the region of the PMF-1 protein that was mutated m pACT 2/PMF-lMu is not responsible for the association of PMF-2 with Nrf-2

Given that PMF-1 contains two coiled-coil regions, one m the ammo terminus ranging from ammo acids 40-88 and a larger one m the carboxy terminus ranging from ammo acids 88-165, two chimenc activation plasmids were constructed. The first one contained the PMF-1 ammo acids 1-88 (pACT2/PMF-lN) and the second contained the PMF- 1 ammo acids 88- 165 (pACT2/PMF- 1 C) Only the pACT2/PMF- 1 C construct (not self-activating) was capable of inducing the transcnption of the reporter genes when co- transforming yeast with pAS2.1 /Nrf-2-LZ These results suggested that the carboxy terminal coiled-coil region of PMF-1 was responsible for interacting with Nrf-2.

In order to confirm the above results, two mutants interrupting the coiled-coil structure m two locations were constructed The first construct, pACT2/PMF-lC-Mul , reduced the coiled-coil region from ammo acids 100-160 to ammo acids 125-160. The second construct, pACT2/PMF- 1 C-Mu2, reduced the coiled-coil region to ammo acids 100- 138 Using the yeast two-hybnd assay, only the wild- type PMF- 1 C construct was capable of activating the reporter genes when co-transforming yeast with pAS2.1/Nrf2- LZ These results indicate that the entire carboxy terminal coiled-coil region of PMF-1 is necessary for the trans-activation of Nrf-2 by PMF-1 Analysis of the leucine zipper region m the carboxy terminus of Nrf-2 mdicated that it possesses a relatively large region capable of forming a coiled-coil structure that would facilitate an association with PMF- 1 In order to determine whether the coiled-coil domain alone is sufficient for the Nrf-2 interaction with PMF-1, a senes of mutations were made m the leucine zipper region of Nrf-2. Mutations were made to destroy the leucme zipper, while maintaining the coiled-coil structure, and separate mutations were made that maintained the leuzme zipper, while altenng the coiled-coil structure. Since the full-length Nrf-2 is self-activating m the yeast two-hybnd system, as indicated m Example 1, only the carboxy terminal region containing ammo acids 434-589 was used PCR was used to generate point mutations resulting m pAS2.1/Nrf-LZ-Mul Complete sequencing of the mutant confirmed that the second and third (of a total of 7) leucmes m the leucine zipper region were replaced by valmes This results m a loss of the leucme 42 zipper motif while maintaining the coiled-coil structure When compared to the wild- type construct, pAS2 1/Nrf-LZ, the mutant lacking the leucine zipper motif was unable to associate with PMF- 1 and activate transcnption m the yeast two-hybnd system These results indicate that the leucme zipper motif m Nrf-2 is required for Nrf-2/PMF-l bmdmg However, the results do not conclusively establish whether or not the coiled-coil motif is also required, since the ammo acid replacement did not significantly alter the coiled-coil domain

In order to determine whether or not the coiled-coil motif is also required, an attempt was made to interrupt the coiled-coil structure while leaving the leucine zipper motif mtact In order to accomplish this, two mutations were made m the Nrf-2-LZ construct (pAS2 l/Nfr-2-LZ-Mu3 (Leu 524- Pro) and pAS2 l/Nrf-2-LZ-Mu4 (Lys 517→Pro)) using prolme to interrupt the coiled-coil structure Although neither of the mutant constructs changed any of the leucmes required for the leucme zipper motif, neither construct was capable of interacting with PMF-1 Although these results suggest that both of the leucine zipper motif and the coiled-coil structure are required for Nrf- 2/PMF-l interaction, an alternative mteφretation is that the prolme substitutions not only interrupted the coiled-coil structure, but also distorted the coiled structure of the leucine zipper to a significant extent This potential change m leucine zipper structure may be responsible for the lack of Nrf-2/PMF-l interaction, rather than the sole loss of the coiled- coil structure

All of the references cited herein, including patents, patent applications, and publications, are hereby incoφorated in their entireties by reference

While this invention has been descnbed with an emphasis upon preferred embodiments, it will be obvious to those of ordmary skill in the art that vanations of the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically descnbed herein. Accordingly, this mvention mcludes all modifications encompassed within the spmt and scope of the invention as defined by the following claims

Claims

43WHAT IS CLAIMED IS:

1. An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding human polyamine-modulated factor- 1 (PMF-1) or a fragment thereof

2. The isolated or purified nucleic acid molecule of claim 1 , which (i) encodes the amino acid sequence of SEQ ID NO: 2 or a fragment thereof comprising at least 17 nucleotides, (ii) consists essentially of the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof comprising at least 17 nucleotides, (iii) hybridizes under low stringency conditions to an isolated or purified nucleic acid molecule consisting essentially of the nucleotide sequence that is complementary to SEQ ID NO: 1 or a fragment thereof, or (iv) shares 40% or more identity with SEQ ID NO: 1.

3. An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a variant PMF-1, which comprises one or more insertions, deletions and/or substitutions, wherein the variant PMF-1 encoded by said isolated or purified nucleic acid molecule does not differ functionally from the corresponding unmodified PMF-1, or a fragment thereof comprising at least 17 nucleotides.

4. The isolated or purified nucleic acid molecule of claim 3, wherein the variant PMF-1 increases the transcription of the gene encoding SSAT at least about 90% as well as the unmodified PMF-1 comprising SEQ ID NO: 2 as determined by in vitro assay in the presence of excess Nrf-2.

5. The isolated or purified nucleic acid molecule of claim 3, wherein said one or more substitution(s) do(es) not result in a change in an amino acid of the encoded PMF-1 or results in the substitution of an amino acid of the encoded PMF-1 with another amino acid of approximately equivalent size, shape and charge.

6. An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to a nucleotide sequence encoding human PMF- 1 or a fragment thereof.

7. The isolated or purified nucleic acid molecule of claim 6, which (I) is complementary to a nucleotide sequence encoding the ammo acid sequence of SEQ ID NO 2 or a fragment thereof compnsing at least 17 nucleotides, (n) is complementary to the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof comprising at least 17 nucleotides, (in) hybridizes under low stringency conditions to an isolated or punfied nucleic acid molecule consisting essentially of SEQ ID NO: 1 or a fragment thereof, or (iv) shares 40% or more identity with the nucleotide sequence that is complementary to SEQ ID NO: 1 or a fragment thereof.

8. An isolated or purified nucleic acid molecule consisting essentially of a nucleotide sequence that is complementary to either of a nucleotide sequence encoding a vanant PMF-1 or a fragment thereof compnsing at least 17 nucleotides.

9. A vector comprising the isolated or purified nucleic acid molecule of any of claims 1-8.

10. A composition compnsmg the isolated or purified nucleic acid molecule of any of claims 1-8 and a pharmaceutically acceptable carrier.

11. A composition compnsmg the vector of claim 9 and a pharmaceutically acceptable carrier.

12. A cell compnsing the vector of claim 9.

13. An isolated or purified polypeptide molecule consisting essentially of an ammo acid sequence encoding human PMF-1 or a fragment thereof, either one of which is optionally glycoslyated, amidated, carboxylated, phosphorylated, estenfied, N- acylated or converted into an acid addition salt

14 The isolated or purified polypeptide molecule of claim 13, which (I) consists essentially of the ammo acid sequence of SEQ ID NO 2 or a fragment thereof comprising at least 8 armno acids or (n) shares 40% or more identity with SEQ ID NO 2 or a fragment thereof

15 An isolated or purified polypeptide molecule consisting essentially of an ammo acid sequence encoding a variant PMF- 1 or a fragment thereof compnsing at least 8 ammo acids, either one of which is optionally glycoslyated, amidated, carboxylated, phosphorylated, estenfied, N-acylated or converted into an acid addition salt

16 A composition comprising the isolated or purified polypeptide molecule of any of claims 13-15 and a pharmaceutically acceptable carrier.

17 A conjugate compnsing the isolated or purified polypeptide molecule of any of claims 13-15 and a targeting moiety

18 The conjugate of claim 17, wherein said targeting moiety is an antibody or an antigemcally reactive fragment thereof

19 A hybridoma cell line that produces a monoclonal antibody that is specific for the isolated or punfied polypeptide molecule of any of claims 13-15

20 The monoclonal antibody produced by the hybridoma cell line of claim 19

21 A polyclonal antiserum raised agamst the isolated or purified polypeptide molecule of any of claims 13-15 46

22. A method of regulating a PMF- 1 -responsive gene in a cell, which method comprises contacting said cell with a gene-regulating amount of PMF-1, whereupon said PMF-1 -responsive gene in said cell is regulated.

23. The method of claim 22, wherein said PMF- 1 -responsive gene encodes spermidine/spermine N'-acetyltransferase (SSAT).

24. The method of claim 22 or 23, wherein PMF-1 regulates the PMF-1- responsive gene by increasing the transcription of the gene.

25. The method of any of claims 22-24, wherein the cell is contacted with PMF-1 by contacting the cell with an isolated or purified nucleic acid molecule which consists essentially of a nucleotide sequence which encodes PMF- 1 or a functional fragment thereof and which is operably linked to a promoter, wherein said isolated or purified nucleic acid molecule is optionally in the form of a vector.

26. The method of claim 25, wherein the isolated or purified nucleic acid molecule (i) encodes the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, (ii) consists essentially of the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof, (iii) hybridizes under low stringency conditions to an isolated or purified nucleic acid molecule consisting essentially of the nucleotide sequence that is complementary to SEQ ID NO: 1 or a fragment thereof, or (iv) shares 40% or more identity with SEQ ID NO: 1.

27. The method of any of claims 22-24, wherein the cell is contacted with PMF- 1 by contacting the cell with an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding PMF-1 or a fragment thereof.

28. The method of claim 27, wherein the isolated or purified polypeptide molecule either (i) consists essentially of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof or (ii) shares 40% or more identity with SEQ ID NO: 2 or a fragment thereof.

29. The method of claim 27 or 28, wherein the isolated or purified polypeptide molecule is contained within a liposome comprising a cell-surface targeting moiety that binds to the cell being contacted.

30. The method of any of claims 22-24, wherein the cell is contacted with

PMF-1 by contacting the cell with a conjugate comprising an isolated or purified polypeptide molecule consisting essentially of an amino acid sequence encoding PMF- 1 or a fragment thereof and a cell-surface targeting moiety that binds to the cell being contacted.

31. The method of claim 30, wherein the isolated or purified polypeptide molecule either (i) consists essentially of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof or (ii) shares 40% or more identity with SEQ ID NO: 2 or a fragment thereof.

32. The method of any of claims 22-31 , wherein the cell is a cancerous cell and the viability and/or metastatic potential of the cancerous cell is decreased.

33. The method of claim 32, which method further comprises administering an anti-cancer agent.

34. The method of claim 33, wherein the anti-cancer agent is a polyamine or an analogue thereof.

35. A method of assessing the sensitivity of cells to treatment with a polyamine or an analogue thereof, which method comprises assessing the level of 48 PMF-1 mRNA or PMF-1 polypeptide in a sample of the cells after contact with a polyamine or an analogue thereof, wherein an increase in the level of PMF- 1 mRNA or PMF- 1 polypeptide in the sample of cells after contact with a polyamine or an analogue thereof as compared to a control sample or a sample of the cells before contact with the polyamine or analogue thereof is indicative of sensitivity of the cells to treatment with a polyamine or an analogue thereof.

36. The method of claim 35, wherein said cells are cancerous.

37. A method of assessing the effectiveness of treatment of cells with a polyamine or an analogue thereof, which method comprises assessing the level of PMF-1 mRNA or PMF-1 polypeptide in a sample of the cells before and during treatment of the cells with a polyamine or an analogue thereof, wherein an increase in the level of PMF-1 mRNA or PMF-1 polypeptide in the cells during freatment of the cells with a polyamine or an analogue thereof is indicative of efficacy of treatment of the cells with a polyamine or an analogue thereof.

38. The method of claim 37, wherein said cells are cancerous.

39. An isolated and purified DNA molecule consisting essentially of the genomic sequence of human PMF-1 or a fragment thereof.

40. The isolated or purified DNA molecule of claim 39, which consists essentially of the nucleotide sequence of SEQ ID NO: 3 or a fragment thereof of at least 16 nucleotides.