CA2663498A1 - Compositions and methods to prevent cancer with cupredoxins - Google Patents

Abstract

The present invention relates to compositions comprising peptides that may be variants, derivatives and structural equivalents of cupredoxins that inhibit the development of premalignant lesions in mammalian cells, tissues and animals. Specifically, these compositions may comprise azurin from Pseudomonas aeruginosa, and/or the 50-77 residue region of azurin (p28). The present invention further relates to compositions that may comprise cupredoxin(s), and/or variants, derivatives or structural equivalents of cupredoxins, that retain the ability to inhibit the development of premalignant lesions in mammalian cells, tissues or animals. These compositions may be peptides or pharmaceutical compositions, among others. The compositions of the invention may be used to prevent the development of premalignant lesions in mammalian cells, tissues and animals, and thus prevent cancer.

Description

COMPOSITIONS AND METHODS TO PREVENT CANCER WITH CtiPREDOXINS
CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 L.S.C. 119 and 120, of Provisional U.S.
Application Serial No, 60,`844,358, filed September 14, 2006, which claims prioritv to U.S.
Patent Application No. U.S. Patent Application No. 11/244,105, fited October 6, 2005, which claims priority to U.S. Provisional Patent Application Serial No. 60/616,782, filed October 7, 2004, and U.S. Provisional Patent Application Serial No. 60,"680,500_ filed May 13, 2005, and is a continuation-in-part of U.S. Patent Application Serial Number 10.'7120,603, filed November 11, 2003. which claims priority to U.S. Provisional Patent Application Serial No.
60/414,550, filed August 15, 2003, and which is a continuation-in-part of U.S.
Patent Application Serial Number 10r047,710, filed January 15, 2002, which claims priority to U.S.
Provisional Patent Application Serial Number 60/269,133, filed February 15, 2001. The entire content of these prior applications is fully incorporated herein by reference.
STATEMENT OF GOVERNMENTAL INTEREST
The subject matter of this application has been supported by research gants from the National I.nstitutes of.Health (NIH), Bethesda, Maryland, U.S.A., (Grant Numbers Al 16790-21. ES 04050-16, A.I 45541, CA09432 and N01-CM97567). The government mav have certain rights in this invention.

FIELD OF THE INVENTION
The present invention relates to compositions comprising variants, derivatives and structural equivalents of cupredoxins that inhibit the development of premalignant lesions in mammalian cells, tissues and anirnals. The invention also relates to the use of cupredoxins, and va.riants, derivatives and struc,turally equivalents of cupredoxins, as chc~~-nopreventive mammals to inhibit tbÃ, ~c~. C'Ã=pm.cnt of premalignant lesions, and ultimately cancer.
BACKGROUND
Cancer chemoprevention is the use of natural, synthetic or biologic chemical agents to revCrse, suppress, or prevent carc.-ino~enie progression ti~ invasive cancer.
Recent clinical in preventiiig cancer in high-risk populations that chetnopreventive therapy is a generalized carcinogen exposure throughout the tissue field results in diffuse cpithelial irljury in tissue and clonal proliferation of the mutated cells. These genetic mutations throughout the field increase the likelihood that one or more premalignant or malignant lesions mav develop in the tield. 11?Iultistep carcinogenesis in the stepwise accumulation of these genetic and phenotypic alterations. Arresting one or more steps in the multistep cancinogenesis may impede or prevent the development of cancer. See generallv Tsao et al., CA
Cancer J Clin 54:1. SO-1 80 (2004).

Aaurin, and other cupredoxins, are cytotoxic specificallv towards cancer cells. Azurin induces apoptosis in J7714 lung cancer cells. Yamada et al., PNAS 99(22):14098-(2002). On entry into .I774 lung cancer cells, azurin localizes in the cytosol and nuclear fractions, and forms a complex with tumor suppressor protein p53, thereby stabilizing it and enhancing its intracellular level. Id. The induction of azurin-mediated apoptosis is not limited to J774 cells. Azurin can also enter cancer cells such as human melanoma UISO-Mel-2 or human breast cancer MCF-7 cells. Yamada et al_. Infect Immun. 70:7054-(2002); Punj et al., Oncogene. 23:2367-2378 (2004). In both cases, azurin allowed the elevation of the intracellular p53 levels, leading to enhanced Bax formation and induction of apoptosis in such cells. Most interestingly, intraperitoneal injection of azurin in nude tnice harboring xenografted Mel-2 or iVICF-7human cancers led to statistically significant regression of such cancers. Id.
The mouse mammary gland organ culture (MMOC) assay may be used to evaluate the inhibitory effects of potential chernopreventive agents on both hormone-induced structural differentiation of mammary glands and on the development of DMBA-induced preneoplastic hvperplastic alveolar Dodule-like lesions in the gland. Mammary glands from young, virgin animals, when incubated for 6 days in the presence of insulin (I) =- prolactin (P) - aldosterone.
(A), cari into fut.lyr-gr< ,1:i glands. These glands morpholoically resemble the glands obtaiiaed fi-om. pregnant mice. Ald;_-terone: can be rcl,':.-i.E3d bv (E) progesterone. (Pg) Inclusion of hydroc~ (H) to the mc;diam stiniular~.~, the functional differentiation of the mammary glands. ~,I.::-hta a7d Banerjee, Acta Endoe.rinol. 80:501 (19=5)g M.ehta and Moon, Breast C=ancer: 1'reatrrtent andPragnasu 30{}, 300 (Basil A Stoll ecl., Bl:rcl_,;. I11 Prv=:, 1~~~6). Thus, the ht : r~~ ie ir;c 1 t,~ StruCtu-:~I an": -.iinctior~al observed . i . . . . i. ' . ~ f ~ l .1.: . ~ ,. .

Mice exhibit a distinct preneoplastic stage prior to canctr formatian in MMOC.
Such preneoplastic lesions in C3H mice are induced by murine mammary tumor virus or in BALBf'c mice by DMBA. Exposure of the glands to2pg,`rnl DMBA bet~veen days 3 and 4 ot grawth phases followed by regression of the glands for 2-3 -vveeks in the medium containing only insulin, results in the formation of mammary alveolar lesions (MAL).
Hawthame et al., Ph.armaceutical Biology 40:70-74 (2002); Mehta et al., Methods in Cell Science 19:19-1-4 (139i). Furthermore, transplantation of epithelial cells.
prepared from glands containing the DMBA-induced mammary lesions, into svngeneic host resulted in the development of mammary adenocarcinoma. Telang et al., PNAS 76:5886-5890 (1979).
Pathologically, these tumors were similar to those obsenied in vivo when mice of the same strain are administered DMBA. Id.

DMBA-induced mammary lesion formation in ilvL'VIOC can be inhibited by avari.ety of classes of chemopreventive agents such as retinoids. These agents include chemopreventive agents derived from the natural products such as brassinin and resveretrol, thiols, antioxidants, inhibitors of ornithine decarboxylase such as OFMO and deguelin, inhibitors of prostaglandin synthesis, Ca regulators, etc.. Jang et al., Science 275:218-220 (1997); Mehta, Eur. J. Cancer 36:1275-I282 (2000); Metha et al., J. Natl.
Cancer Inst.
89:212-219 (1997). These studies clearly demonstrate that this organ culture svstem offers a unique model to determine the effectiveness of compounds against mammary carcinogenesis.
The results can be expected to closely correlate to the inhibition obtained by in vivo administration of such compounds.

T'he MMOC may also be induced to farm mammary ductal lesions (MDL). The MDL
can be induced if estrogen and progesterone instead of aldosterone and hydrocortisone are included in the medium. The alveolar structures in the presence ofcsva.rian steroids are very sma.lllaut the intraductal lesicins are observed in histopathological sections. Mehta et al., J.
Natl. Cancer Inst. 93:1103-1106 (2001). `Ih(, i."~s~ ~~i ~,, Nk',icb selectively work on ovarian bonnone dependent ER+ breast ta nt%,._i~_i., inhibited MDL formation and. not MA.L. Thus, this modified culture model in addition to conventiotial MAL I.nduction.
protocol now can be used to evaluate effects of chemopreventive agents on both MAL. and MOL.
., -~ . .., ._. . ~.. . .~!..,.. ~_ _ . .. .__ .1t . i r~~ ~Ã . . . . ._ i}f sion.s. ~,-ach initial development of premalignant lesions, induce cell death in premalignant lesions that forin. and;'or prevent the development of premalignant lesions into malignant lesions. Such chemopreventive agents would have great utility in treating, in particular, patients who are at a high risk of developing cancer, due to either the presence of higb.-risk features, the presence of pre-malignant lesions, or the previous of cancer or premalignant lesions.
Sti MMARY OF THE EMBODIMENTS
The present invention relates to compositions comprzsing peptides that may be variants, derivatives and structural equivalents of cupredoxins that inhibit the development of premalignant lesions in mammalian cells, tissues and animals. Specifically, these compositipn.s may comprisc azurin from Pseudanionus aeruginosa, and/or the 50-77 residue region of azurin (p28). The present invention further relates to compositions that may coinprise cupredoxin(s), and:`or variants, derivatives or structural equivalents of cupredoxins, that retain the ability to inhibit the development of premalignant lesions in mammalian cells, tissues or animals. These compositions may be isolated peptides or pharmaceutical compositions, among others. The compositions of the invention may be used in methods to prevent the development of cancer in mammalian patients.

One aspect of the invention are isolated peptides that may be a variant, derivative or structural equivalent of a cupredoxin; and may inhibit the development of premalignant lesions in rrlainmalian tissue. The cupredoxin may be azurin, pseudoazurin, plastocyanin, rusticyanin, Laz, auracyanin, stellacyanin and cucumber basic protein, and specifically may be azurin. The cupredoxin may be from an organism strch as Pseudomonas aerliginosca, Alcalagenes faeccrlis, Ulva pertussis, Aeltronacsbacter xylasoxidan, Bordetella bronchiseptica, I1-fethIvlomonas sp., Neisseria nieningitidis, Xeisseria gonorrhea, Pseaidamotiasfluoresc~.~ns, Pseudomonas chlororaplzis, Xvlella.fastidiosa and Vihr=ifl parahactnoIyticus, and specifically may be Pseuaoj~; ,: c{ rugirosa. In some embodiments, the peptide may be part of SEQ ID
NOS: 1, 3-19, or has at least 80% amino acid sequence identity to SEQ ID NOS:
i, 3-19.
In some ernbodiments, the isolated peptide may be a truncation of a crzpred.oxin. The isolated peptide may be more than about. 10 residues and not more than about 100 residues.
The isolr.ted pcptid.c may comprise, or,-Atcrnatively consi~;t <-ff.
~~eudramonas arruginosa Another aspect of the inventidti is a pharmaceutical composition that may comprise at least one, or two, cupredoxins or isolated peptides of the invention in a pharmaceutically acceptable carrier. ne pharnnaceutical composition znalv be formulated for intravenous administration. In some embodiments, the cupredoxin in the pharrma.ceutical composition may be from an organism such as Psczidonaonas aer~uginosa, U'Ivcr pertussis, Alcaligenes . faecalis, Achrotnobactcr x;losoxidan, Bordet~,~lla bronchiseptica, .1/lothYlomonas sp..
A`eisseria rrieningitidis, ;Veisscria gonorrhea, Pseudonaonqsfluorescens, Pseudornonas chlororaphfs, Xvlellca fastidiosa and Vibrio parcxhaemolytacus, and specificallv may be from Pseudomonas aeruginosa. The cupredoxin may be SEQ TD'~~QS: 1, 3-19.
Another aspect of the invention is a method to treat a matnmalian patient by administering to the patient a therapeutically effective amount of the pharmaceutical composition of the invention. The patient may be human, and may be at a higher risk to develop cancer than the general population. In some embodiments, the cancer may be melanoma, breast, pancreas, glioblastoma, astrocyloma, lung, colorectal, neck and head, bladder, prostate, skin, or cervical cancer. In some embodiments, the patient may have at least one high risk feature, prernalignant lesions or have been cured of cancer or premalignant lesions.
The pharmaceutical composition may be admillistered by intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, vitreous injection or oral, and specifically may be administered bv intravenous injection. The pharmaceutical composition may be co-administered with at least one other chemopreventive drug, and specifically at about the same time as another chemopreventive drug.
Another aspect of the invention is a kit comprising the pharmaceutical composition of ?5 the i.::1, c: aionin avial. The kit mav be designed for intravenous administration.
Another aspect of the invention is a method to study the d: Ã,-~pn-aent of cancer comprising contacting i-na-rrmalian cells with a cuprecloxin. or peptide of the invention and measuring the development of prcrnalignant and nialignant cells. In some embodiments, the cells may be hurrian andior marnmar~- cells. In some embodiments, the cells are induced to develop prenialignant lesior~-,.
A.__ pec _ aS ara exPf . encodE~ ~)f These and other aspects. advantages, and features of the. invention will become apparent from the following figures and detailed description of the specific embodiments.

BRIEF DESCRIPTION OF THE SEQUENCES
SEQ fD NO: 1. Amino acid sequence of azurin from Pseacdoraaranas aeru~qinusa.

SEQ ID NO: 2. Amino acid sequence of p28, Pseudcsrnoncas ctc;rtiginosa azurin residues 50-77.
SEQ ID NO: 3. Amino acid sequence of plasfic?cyanin from Phorniidium laminosum.
SEQ ID NO: 4. Amino acid sequence of rusticyanin from 7hiobacillus ferrooxidans.
SEQ ID NO: 5. Amino acid sequence of pseudoazurin from Achrontabaeter cycloclastes.
SEQ ID NO: 6. Amino acid sequence of azurin from Alcaligenes faecalis.
SEQ tD NO: 7. Amino acid sequence of azurin from Ahromobacter xylosoxidans ssp.
denitrificans I.
SEQ ID NO: S. Amino acid sequence of azurin from Bordetella bronchiseptica.
SEQ ID NO: 9. Amino acid sequence of azurin frorn .,Wethylornoraas sp. J.
SEQ ID NO: 10. Amino acid sequence of azurin from 1Veisseria nzeningitidis Z2491.
SEQ ID NO: 11. Amino acid sequence of azurin from Pseudomonas fluorescen.
SEQ ID NO: 12. Amino acid sequence of azurin from. Pseudomonas c]zlororaphis.
SEQ ID NO: 13. Amino acid sequence of azurin from Xylella.fastidiosa 9a5c.
SEQ ID NO: 14. Amino acid sequence of stellacyanin from Cucumis sativus.
SEQ ID NO: 15, Amino acid sequence of auracyanin A from Chlor Jlexus aurdntiacacs.
SEQ ID NO: 16. Amino acid sequence of auracyanin B from Chloro.flexus aurantiacus.
SEQ ID NO: 17. Amino acid sequence of cucumber basic protein from Cucumis sativus.
SEQ ID NO: IS. Amino acid sequence of Laz from J'V'eisserid gonorrhocae F62.

SEQ ID NO: 19. Amitio acid sequence ~.~f the azurin from i'ibrio parahcaenioly>ticus.
SEQ ID NO: 20. Amino acid sequence c, i: i,o acids 571 to 89 of a.l.. ~:-:l lin B of Citlorqflexus aurantiacus.
SEQ LD NO: 21. Amino acid sequence of amino acids 51-77 ofT-'r,,tdorn~ncis syringae azunn.
SEQ ID NO: 22, Amino acid sequence of amino acids 89-11 y of Neisseria meningaizidts Laz.
SI ID NO: 23. V ce acids 52-75 of Vibriop o, SEQ ID NO: 24. Amino acid sequence of axnitto acids 51-77 of Bordetella bronchiseptica azurin.

BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Figure I depicts photographs of all of the glands evaluated for the efficacy ofp2s and azurin. Figure IA shows a representative photograph of alveolar lesions in a DMBA-treated gland and its comparison with a gland that was treated with DMBA along with a chem opreventi4--e agent. Figures 1 B-1 G show representative photographs of the effects of p28 on the development of alveolar lesions.
Figure 2. Figure 2 depicts a graph showing the efficacy of p28 against DMBA-induced mammary alveolar lesions.
Figure 3. Figure 3 depicts photographs of representative sections of ductal lesions and effect of p28.
Figure 4. Figure 4 depicts a graph showing the efficacy of p28 against DMBA-induced ductal lesions DETAILED DESCRIPTION OF THE INVENTION
Definitions As used herein, the teron "cell" includes either the singular or the plural of the term, unless specifically described as a"singie cell."
As used herein, the terms "poiypeptide,," "peptide," and "protein" are used interchangeably to refer to apolymer of amino acid residues. The terms apply to amino acid polymers in which one or n-iore amino acid residuc is an artificial chemical analogue of a corresponding naturally occurring amino acid. The terms also apply to naturally occurring amino acid polymers. The terzns "polvpeptidc," õpeptide," and "protein" are also inclusive of modifications inÃ;lod:r,~;, but not iimited. to, ~~,Iyc~~s~:'a tior~, lipid attachment, s~alfa.tiori, gamsna-carhoxyla.tion of Lic acid residues, hydro-,~ .; , : , ~n t:nd ADP-ribosyla.tiors. l. _; b,,-appreciated that pol~p~, ptides are not alwavs entir(;,,. linear. For instance, polypep`io:;s rnay be branched as a result of ubiquitination and they may be circular (with or without branchirzg} generally as a result of pw -translation events, including natural processing event and events broutlit about bv hUMF:Ãj ~.. ~rula.tiorz which do not occur nat-urallv, Circular, nc~r~- riatUral . cl..

As used herein, the term "pbarmacoloLic activity" means the effect of a drug or other chemical on a biological system. The effect of chemical may be beneficial (therapeutic) or harrnful (toxic). 'jhe pure chemicals or mixtures may be of natural origin (plant, animal, or mineral) or may be synthetic compounds.
As used herein, the term "premalignant" means precancerous, or before abnormal cells divide without control.
As used herein, the term "lesion" means an area of abnormal tissue.
As used herein, the tert7-i "pathological condition" includes anatomic and physiological deviations from the normal that constitute an impairment of the norrnal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions, and is a response to various factors (as malnutrition, industrial hazards, or climate), to specific infective agents (as worms, parasitic protozoa, bacteria, or viruses), to inherent defects of the organism (as genetic anomalies), or to combinations of these factors.
As used h~,~rein, the term "condition" includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performan.ce of the bodily functions.
As used herein, the term "suffering from" includes presently exhibiting the symptoms of a pathological condition, having a pathological condition even without observable symptoms, in recovery from a pathological condition, or recovered from a pathological condition.

As used herein, the term "chemoprevention" is the use of drugs, vitamins, or other agents to try to reduce the risk of, or delay the development or recurrence of, cancer.
A used herein, the term "treatment" includes preventing, lowering, stopping, or reversing the progression or severity of the condition or symptoms associated with a condition being treated. As such, the term "treatment" includes medical, thc.
=-pLutic:, and;or prophylactic administration, as appropriate. Treatment tnav also include preventing or lessening the developrnent of a condition, such as cancer.
As used herein, the terrn "inhibit cÃ;Il g-owth" rnea.ns the slowing or ceasing of cell division arid/or cell expansion. This term also includes the inhibition of cell development or eeli d,:ath.

a particular condition for which the subject being treated. Detcrmination of a the.rapeuticallv effective amount is well within the capabilitv of those skilled in the art.
The term "substantially pure," as used herein, when used to modify a protein or other cellular product of the invention, refers to. for example. a protein isolated from the growth medium or cellular contents, in a form substantially free of, or unadulterated by, other proteins and:jor other compounds. The term "substantially pure"refers to a factor in an amount of at least about "5%, by dry weight, of isolated f-actiozr, or at least "75%
substantially pure." More specificallv, the terrn "substantially pure" refers to a compound of at least about 85%, bv dry weight, of isolated fraction, or at least "85%
substantially pure."
Most specifically, the term "substantially pure" refers to a compound of at least about 95%, bv dry weight, of isolated fraction, or at least "95% o substantially pure."
The term "substantially pure" may also be used to modify a synthetically-made protein or compound of the invention, where, for example, the synthetic protein is isolated from the reagents and by-products of the synthesis reaction(s).
The terrn "pharmaceutical grade," as used herein, when referring to a peptide or compound of the invention, is a peptide or compound that is isolated substantially or essentially from components which normally accompany the material as it is found in its natural state, including synthesis reagents and by-products, and substantially or essentially isolated from components that would impair its use as a ph.artnaceutical. For example, a "pharrra.aceutical grade" peptide may be isolated from any carcinogen. In some instances, "pharmaceutical grade" may be modified by the intended method of administration, such as "intravenous pharmaceutical grade," in order to specify a peptide or compound that is substantially or essentially isolated from any substance that would render the composition unsuitable for intravenous administration to a patient. For example, an "intravenous pharrrtaecutical grade" pept.i.de may be isolated from detergents, such as S.DS, and an.ti-baeterial agents, such as azide.
Ihe terms f'Isolatccl," "purified" or ": x> 11:;,g1c,llly pure" refer to material '. hich is substantially or essentially free from corza:poner: swbicb nom-tally accompany the n-iaterial as it is found in its native state. Tllus, isolated peptides in accordance with the invention prc-r-.bly do not con :'n rtorrn~-i11 socl d wit i the pcptides in th~:ir in situ An$ inclu6.It:'.
~. .
~~ ; .t [ tri. ta:a~ ~3 :`-' : '1 :;c Ã1~sd~z ~[aaaft tsÃs ~i~x r~t rvtt..
~ts:>t~=tu. t~xà ~

nucleic acid, protein, or respective fragment thereof has been substarrtially removed from its in vivo environment so that it mav be manipulated by the skilled artisa.n, such as but not limited to, nucleotide sequencing, restriction digestion, site-directed mutagenesis, and subeloning into expression vectors for a nucleic acid fragn~.~nt as well as obtaining the protein or protein fragment in substantially pure quantities.
The term "variant" as used herein with respect to a peptide, refers to amino acid sequence variants wbich may have amino acids replaced, deleted, or inserted as compared to the wild-type polypeptide. Variants may be tnmcations of the wild-type peptide. A
"deletion" is the removal of one or more amino acids from witbin the pol.ypeptide, which a "truncation" is the removal of one or more amino acids from one or both ends of the polypeptide. Thus, a variant peptide may be made by manipulation of genes encoding the polypeptide. A variant may be made by altering the basic composition or characteristics of the polypeptide, but not at least some of its pharmacolol;ic activities. For example, a "variant" of azurin can be a mutated azurin that retains its ability to inhibit the development of premalignant mammalian cells. In soine cases, a variant peptide is synthesized witb non-natural amino acids, such as z-(3,5-dinitrobenzoyl)-Lys residues. Ghadiri &
Fernholz, J. Arn.
Chem. Soc., 112:9633-9635 (1990). In some embodiments, the variant has not more than 20 amino acids replaced, deleted or inserted compared to wild-type peptide or part thereof. In some embodiments, the variant has not more than 15 amino acids replaced, deleted or inserted compared to wild-type peptide or part thereof. In some embodiments, the variant has not more than 10 amino acids replaced, deleted or inserted compared to wild-type peptide or part thereof. In some embodiments, the vafiant has not more than 6 amino acids replaced, deleted or inserted compared to wild-type peptide or part thereof. In some embodiments, the variant has not more than 5 amino acids replaced, deleted or inserte-d compared to wild-type peptide or part thereof. In some embodiments, the variant has not more than 3 amino acids replaced, deleted or inserted compared t~~ .vi1d-type peptide or part tb~,~c:of.
Tlic tti,-m "amino acid," as used ineans an ammo acid moiety that comprises arzy1 natu--ally-occurring or non-naturallyr occurring or synthetic amino acid residue. i.e., any moiety comprising at least one carboxyl and at least one amino residue directlv linked by one.

two thre,e, or rriore carbon a:orns. typically one (a) carbon atom.
. . e t er I. : , i ~ . .. i c.
,.._. , _. . . _.l .'~ s . .~~. . .
i peptide such that the peptide still retains some nfits fundamental activities.
For example. a "derivative" of arurin can, for example, be a chemically modified azurin that retains its ability to inhibit angiogenesis in mammalian cells. Chemical xnoditicatians of interest include, but are not limited to, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation or glycosylation of the peptide. In addition, a derivative peptide may be a fusion of a polypeptide or fragment thereof to a chemical compound, such as but not limited to, another peptide, drug molecule or other therapeutic or pharmaceutical agent or a detectable probe.
The term "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a polypeptide that are identical with amino acid residues in a candidate sequence when the two sequences are aligned. To deterrmitie % amino acid identity, sequences are aligned and if necessary, gaps are introduced to achieve the rnaximum %
sequence identity; conservative substitutions are not considered as part of the sequence identity. Amino acid sequence alignment procedures to deternnine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or'Vlegalign (DNASTAR) software is used to aIign, peptide sequences. In a specific embodiment, Blastp (available from the National Center for Biotechnology Information, Bethesda MD) is used using the default pararneters of long complexity filter, expect 10, word size 3, existence 11 and extension 1.
When amino acid sequences are aligned, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B
(which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain %
amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as:
% amino acid sequetlcc identity =_ Xs"Y* 100 where X is the nurnber of amino acid residues scored as identical matches by the, sequence alignment program's or algorithm's alignrnent of A and B and Y is the total number of amino acid residues in B.
1- tbe le:nffl; of alriz :o<d ~:,tFuence A is not equal to the le,ngth of a.millo acid !A denti h~ of A f:, s'r~x~lll the ._ ..:~
;.' 3t~to A.

shorter sequence will be the "B" sequence. For example, when comparing truncated peptides to the corresponding wild-type polypeptide, the truncated peptide will be the``B" sequence.
General The present invention provides compositions comprising cupredoxin, and variants, derivatives and structural equivalents of cupredoxins, and methods to prevent the development of cancer in mami-nals. The invention also provides to variants, derivatives and structural equivalents of cupredoxin that retain the ability to prevent the development of cancer or the re-occurrence of cancer in mammals. Most particularly, the invention provides compositions comprising Pseudom nas aet ziglnosa azurin, variants, derivatives and structural equivalents of azurin, and their use to treat patients, and particularly patients at a higher risk of developing cancer than the general population. Finally, the invention provides methods to study the development of cancer in mammalian cells, tissues and animals by contacting the cells with a cupredoxin, or variant, derivative or structural equivalent thereof, before or after inducing premalignant lesions, and observing the development of premalignant and/or malignant cells.

Previously, it was know that a redox protein elaborated by Pseudomonas aerugisnosa, the cupredoxin azurin, selectively enters J774 lung cancer cells but not normal cells, and induces apoptosis. Zaborina et al., Microbiology 146:252I-2530 (2000). Azurin can also selectively enter and kill human melanoma UISta-Mel-2 or human breast cancer cells. Yamada et al., PNAS 99:I4098-14103 (2002); Punj etetl., Oncogene 23:2367-2378 (2004). Azurin from P. aeruginosa preferentially enters J774 rrrurine reticulum cell sarcoma cells, forms a complex with and stabilizes the tumor suppressor protein p53, enhances the intracellular concentration of p53, and induces apoptosis. Yamada et aL, Infection and linmunity 70:7054-7062 (2002 j. Detailed studies of various domains of the azurin molecule shcJi,), ,:d 'i-:; ::-nino acids 50-77 (p28) (SEQ ID NO: 2) represented a protein transductiOn domain (PTD) critical for internalization and subsequent apoptotic activity.
Yamada ei al., Cell. Micrt-ybial. 7:1418-I431 (2005).

It is ilow known that alurin, and peptides derived from azurirl, such as p2s, havc, cheznopre~=entivc properties. It is now known thr:* a=r-urirr; and p',S;
prc;vÃ-;nt to format.on of prc.`; ;:it pret~ ; : t I_ ,:. -11 a .k ~, .: :;' . . . ...,. ~ " . . flnh of alveolar lesions bv 67%. Likewise, p28 at 25 pg-`ml was found to inhibit the formation of alveolar lesions bv 67%. See Example 1. Further, azurin at 50 ~ugiml was fotu-ici to inhibit the formation of ductal lesions by 7/9 %fl. and p28 at 25 qgr`mI inhibited the formation of ductal lesions by 71%. See Example I. Confocal microscopy and FAC showed that azurin and p28 entered normal murine mammary epithelial cells (MM3MG) and rr7amrnary cancer cells (4T1). P28 also entered human umbilical vein endothelial cells (HUVEC) in a temperature, time and concentration deperrd.ent rnanner and inhibited capillary tube formation of HUVEC
plated on iifiatrigelj~'in a dose dependent manner. It is therefore now known that azurin and variants of azurin may be used to inhibit the fornnation of premalignant preneoplastic lesions, and thus the development of cancer, and specifically breast cancer, in mammalian patients.
Due to the high degree of structural similarity between cupredoxins, it is likely that other cupredoxins will inhibit the formation of premalignant lesions in mammals as well as acurin. Such cupredoxins may be found in, for example, bacteria or plants.
Several cupredoxins are known to have pharnnacokinetic activities similar to those of azurin from Pseudomonas aet-uginosra. For example, rusticyanin from T'6aiobacillus fer-rooxidcrns can also enter macrophages and induce apoptosis. Yamada et aL, Cell Cycle 3:1182-1187 (2004);
Yamada et al.. Cell. Micro. 7: I41 8-143 t(2005). Plastocyanin from Phorfnidium laminosurn and pseudoazurin form Achrornobacter cycloclastes also are cytotoxic towards macrophages.
U.S. Pat. Pub. No. 20060040269, published Feb. 23, 2006. It is therefore contemplated that other cupredoxins may be used in the compositions and methods of the invention. Further, variants, derivatives, and structural equivalents of eupredoxins that retain the ability to inhibit the fornation of cancer in mammals may also be used in the compositions and methods of the invention. These variants and derivatives may include, but are not limited to, truneations of a cupredoxizi, conservative substitutions of amino acids and proteins modifications such as FEGyiatic.-~n and all-hydrocarbon stabling of a-he-Iices.
Compositions of the Invention The invention provi.des for peptides that are variants, derivatzves or structural equivalents ofcupredox.i.n that inhibit the development of premalignant lesions in mammalian cel1~, tissues and z nimals. The invention further prc vi;..'e.S For pe,pticlt~s th:.t are v;;iri,mtc:

l." . .fti T u~.'Ia :St -_'~ J _ _ 1 . Ci- In In So, ra some embodiments, the peptide is substantiaily pure or pharmaceutical grade.
In other embodiments, the peptide is in a composition that comprises, or consists essentially of, the peptide. In another specific embodiment, the peptide is non-antigenic and does not raise an immune response in a mammal, and more specifically a human. In some embodiments, the peptide is less that a full-length cupredoxin, and retains some of the pharmacologic activities of the cupredoxins. Specifically, in some embodiments. the peptide may retain the ability to inhibit the development of premalignant lesions in the mouse mammary gland organ culture.
The invention also provides compositions comprising at least one peptide that is a cupredoxin, or variant, derivative or structural equivalent of a cupredoxin, specifically in a pb.arnnaceutical composition. In specific embodiments, the pharmaceutical composition is designed for a particular mode of administration, for example, but not limited to, oral, intraperitoneal, or intravenous. Such compositions may be hydrated in water, or may be dried (such as by lyophilization) for later hydration. Such compositions may be in solvents other than water, such as but not limited to, alcohol.
Because of the high structural homology between the cupredoxins, it is contemplated that cupredoxins will have the same chemopreventive properties as azurin and p28. In some embodiments, the cupredoxin is, but is not limited to, azurin, pseudoazurin, plastocyanin, rusticyanin, auracyanin, stellacyanin, cucumber basic protein or Laz. In particularly specific embodiments, the azurin is derived from Pseudomonas aeruginosa, Alcaligenes f'aecalis, Acht=ornpbacter.xvlosaxidans ssp.denitrificans I, Bordetella bronchiseptica, sVIethylomonas sp., iVeissef ia meningitidis, Neisseria gonorrhea, Psezidomonas fluorescens, Pseudornonas clilor-oraphis, Xylellafiastidiosa, Ulvca pertussis or Yibrio parahaeinolyt/cus. Iti a very specific embodiment, the azurin is from Pseudomonas aeruginosa. In other specific etnbodiinents, the cupredoxin comprises an amino acid seclutnce that is SEQ ID
Nt7; l, 3-19.
15 The invention prcrvicles peptides that are azrzino. L variants which have amino acids replaced, deleted, or inserted as compared to the wild-type cupredoxin. Variants of the invention rnay be truraeations oi"tb.e wild-type eupredoxin. In some embodiments, the peptide of the invention comprises a region of a cuprecioxin tbat is less that the full length, wild-type polypeptide. In some embodiments, the peptide of the invention comprises rrlojre than about 10 morcth. .:~ ,ibow 15 residae- or more tha:i .bout 20 residues of a .. . ..l . ~i.F , 1. `. _ dt .. .. . . . . .._ about 30 residues or not more than about 20 residues of a truncated eupredoxin. In some embodiments. a cupredoxin has to the peptfde, and more specifically SEQ ID
NOS: 1, 3-19 as to the peptide of the invention, at least about '70% amino acid sequence identity, at least about 80% amino acid sequence identity, at least about 90% amino acid sequence identity, at least about 95% amino acid sequence identity or at least about 99% amino acid sequence identitv.
In specific embodiments, the variailt of cupredoxin comprises P. aeruginosa azurin residues 50-7 7(p28, SEQ ID NO: 2), azurin residues 50-67, or azurin residues 36-88. In other embodiments, the variant of cupredoxin consists ofl? rceniginosa azurin residues 50-77, azurin residues 50-67, or azurin residues 36-88. In other specific embodiments, the variant consists of the equivalent residues of a cupredoxin other that azurin.
It is also contemplated that other cupredoxin variants can be designed that have a similar pharmcologic activity to azurin residues 50-77, or azurin residues 36-88. To do tbis, the subject cupredoxin amino acid sequence will be aligned to the Pseudomonas aeruginosa azur~r n sequence using BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), the relevant residues located on the P. aeruginosa azurin amino acid sequence, and the equivalent residues found on the subject cupredoxin sequence, and the equivalent peptide thus desiggried.
In one embodiment of the invention, the cupredoxin variant contains at least amino acids 57 to 89 of auracyanin B of Chloroflexus aurantiacus (SEQ ID NO: 20). In another embodiment of the invention, the cupredoxin variant contains at least amino acids 51-77 of Pseudomonas syringae azurin (SEQ ID NO: 21 ). In another embodiment of the invention, the cupredoxin variant contains at least amino acids 89-115 of.)N'eissericc meningitidis Laz (SEQ ID NO: 22). In another embodiment of the invention, the cupredoxin variant contains at least amino acids 52-78 of Vibrio parcrhaemolvticus azurin (SEQ ID NO: 23).
In another embodiment of the invention, the cupredoxin variant contains at least amino acids 51-7 7 of 2'~ Bordeiclla bronchiseptica azurin (SEQ ID NO: 24).
Tlie va.fiants mav alsi.i include peptides made with synthetiÃ. ::r rio -t'Jds r-rw ~_r_-",, occurring. For exatnple, non-naturally occurring amino acids may be ir>_tegrated into tile variant peptide to extend or optimize the half-life of the cotrtposition in the bloodstream.
Such variants include, but are not limited to, D,L-peptides (diastereorner).
(fbr- example 1t) Futaki ~~t al., J. Biol. Chem. 276t "):5836-4C) (20C11)s Papc~ , ~ar;.:~r Res. 6~(lr';t "~ 86 (2004):
annino acids (fo nc$n-natural amino acid followed by hydrocarbon stapling Vor example Sc-bafrneister etal., J. Am.
Chem. Soc. 122:5891-5892 (2000); Vvalenski et al., Sc,ienee, 305:1466-1470 (2004)), and peptides comprising E-(3,5-diraitrobe-nzoyl)-Lvs residues.
In other embodiments, the peptide ol-thc invcntion is a derivative of a cupredoxin.
The derivatives of cupredoxin are chemical modifications of the peptide such that the peptide still retains some of its fundamental activities. For example, a"derivative"
of azurin can be a chemically modified azurin that retains its ability to inhibit the development of premalignant lesions in mammalian cells, tissues or animals. Chemical modifications of interest include, but are not limited to, hytdrocarbon stabling, amidation, acetylation, sulfation, polyethylene glycol (PEG) rnodification, phosphorylation and glycosylation of the peptide.
In addition, a derivative peptide maybe a fusion of a cupredoxin, or variarrt, derivative or structural equivalent thereof to a chemical compound, such as but not limited to, another peptide, drug molecule or other therapeutic or pharrnaccutical agent or a detectable probe.
Derivatives of interest include chemical modifications by which the half-life in the bloodstream of the peptides and compositions of the invention can be extended or optimized, such as by several methods well known to those in the art, including but not limited to, circularizzed peptides (fot-ex.crrnple Monk et al., BioDrugs 19(4):261-78, (2005); DeFreest et al., J.
Pept. Res.
63(5):409-19 (2004)), N- and C- terminal modifications Vor example Labrie et al., Clin.
Invest. Med. 13(5):275-8, (1990)), and olefin-containing non-natural amino acid followed by hydrocarbon stapling (for example Schafineister etrrl., J. Am. Chem. Soc.
122:5891-5892 (2000); Walenski et ccl., Science 305:1466-1470 (2004)).
In another embodiment, the peptide is a structural equivalent of a cupredoxin.
Examples of studies that determine significant structural homology between cupredoxins and other proteins include Toth et al. (Developmental Cell 1:82-92 (2001)).
Specifically, significant structural homology between. a cupredoxin and the structural equivalent may be determinÃ:d by using the VAST algorithm. Gibrat et aI. Curr Opin Stract Biol 6:31/ 7-385 (1996); Madej e=t al., Proteins 23:356-3690 (1995). In specific embodiments, the VAST p value, ftom a structural comparison of a cupredoxin to the structural equivalent may be less than about 10-3. less than about 1 0-', or less than about 1 0- In other enibodiments, s~~=zilicant struc;ural homologv between a c_,tpredoxin,_;nd the s`ntct~aral erl-aivalent may K
the DALI algorithm, 1 r r<~ c,,:. J. ~:i, mol. 233:123-138 G

In specific embodiments, the DALI Z score for a paircvxse structural eornparison is at least about 3.5, at least about 7.0, or at least about 10Ø

It is contemplated that the peptides of the composition of invention may be more than one of a variant, derivative and/or structural equivalent of a cupredoxin. For example. the peptides may<be a truncation of azurin that has been PEGylated, thus making it both a variant and a derivative. In one embodiment, the peptides of the invention are svnthesized with a,a-disubstituted non-natural amino acids containing olefin-bearing tethers, followed by an all-hydrocarbon "staple" by ruthenium catalyzed olefin metathesis. Scharmeister et aI.,1. .Am.
Chem. Soc. 121-:5$9I-5892 (2000); Walensky et rcl., Science 305:146i6-14 70 (2004).
Additionally, peptides that are structural equivalents of azurin may be fused to other peptides, thus making a peptide that is both a structural equivalent and a derivative.
These examples are merely to illustrate and not to limit the invention. Variants, derivatives or structural equivalents of cupredoxin may or may not bind copper.

In some embodiments, the cupredoxin, or variant, derivative or structural equivalent thereof has some of the pharmaeologic activities of the P. aeruginosa azurin, and specifically p28. In a specific embodiment, the cupredoxins and variants, derivatives and structural equivalents of cupredoxins that may inhibit prevent the development of premalignant lesions in mammalian cells, tissues or animals, and specifically but not limited to, mammary gland cells. The invention also provides for the cupredoxins and variants, derivatives and structural equivalents of cupredoxins that may have the ability to inhibit the development of mammalian premalignant lesions, and specifically but not limited to, melanoma, breast, pancreas, glioblastoma, astrocytoma, lung, colorectal, neck and head, bladder, prostate, skin and cervical cancer cells. Inhibition of the development of cancer cells is any decrease, or lessening of the rate of increase, of the development of premalignant lesions that is 2 55 statistically simificant as compared to c,ontrol trea.ttn.ents.
Because it is now known that cupredoxins can inhibit the development of premalignant lesions and ultimately caiicer in mammalian cells, tissu.es or animals, and specifically breast cells, and more specifically, mouse inarnmary gland cells, it is now possible to design variants and derivatives of cupredoxins that retain this chemopreventive act:~,ity. Sut:hvaria.nts, dcriva'.ves _.nd struc. 1-,i cqaiva.lents can b-rn:,& by, for ex;:npie.

OrucLbiYGt . , . _ .. .ti. ..4Y5(, specifically chemopreventive a.ctivity in the mouse man-irnarv gland organ culture using one of manv methods known in the art, such the exemplary method in Example I. It is contemplated that the resulting variants, derivatives and structural equivalents of cupredoxins with chemopreventive activity may be used in the methods of the invention, in place of or in addition to azurirt or p?-S.
In some specific embodiments, the variant_ derivative or structural equivalent of cupredoxin may inhibit the development of 7,12-dimetbylbenz (a) annthracene (DMBA) induced premalignant lesions in a mouse mammary gland orv-an culture (MMOC) to a degree that is statistically different from a non-treated control. A peptide can be tested for this activity by using the MMOC model system is described in Example 1, or as in Mehta et al. (J
tiatl Cancer Inst 93:1103-1106 (2001)j and Mehta et al. (Meth Cell Sci 19:19-24 (1997)}..
Other methods to determine whether cancer development is inhibited another are well known in the art and may be used as well.
In some specific embodiments, the variant, derivative or structural equivalent of cupredoxin inhibits the development of mammary alveolar lesions (MAL) in the a MMOC
model to a degree that is statistically different from a non-treated control.
In some specific embodiments, the variant, derivative or structural equivalent of cupredoxin inhibits the development of marnmary ductal lesions (MDL) in the a MMOC model to a degree that is statistically different from a non-treated control. A peptide can be tested for these activities by using the MMOC model system induced to form premalignant lesions by DMBA, as described in Example 1. Evaluation of development of premalignant lesions in a MMOC
model system may be determined by morphometic analysis, or histopathological analysis, as provided in Example 1.

Cuprecioxitis I`fi~,~~ blue copper proteins (cuprc,de; 1-1--l!", CIC:~ ~()n tran;.-;r prcteins ( l0-20 kI?a} that participate in bacterial electron transfer c.i_ins or of un.Icnov,n function. The copper ion is soIciv bound by the protein matrix. A special distorted trigonal planar arrantTernent to two histidine and one cysteine Iigands around the copper gives rise to very peculiar electronic properties of the metal site a~d an intense blue color. AL
number of cupredoxins h<. iC

-1~-The cupredoxins in general have a low sequence hom.olog,y but high structural homology. Gough & Clotbia, Structure 12:917-925 (2004); De Rienzo et a1., Protein Science 9:1439-1454(20{1(3}. For example, the amino acid sequence of azurin is 31%
identical to that of auracyanin B. 16.3% to that of rusticyanin, 20.3 % to that of plastocyanin, and 17.3% to that of pseudoazurin. See, Table 1. However, the structural simiiarityof these proteins is more pronounced. The VAST p value for the comparison of the structure of azurin to auracyanin B is 10-14, azurin to rusticvanin is 10-5, azurin to plastocyanin is 10'' and arurin to psuedoazurin is 10-4,1 All of the cupredoxins possess an eight-stranded Greek key beta-barrel or beta-sandwich fold and have a highly conserved site architecture. De Rienzo et aL, Protein Science 9:1439-1454 (2000). A prominent hydrophobic patcb, due to the presence of many long chain aliphatic residues such as methionines and leucines, is present around the copper site in azurins, amicyanins, cyanobacterial plastocyanins, cucumber basic protein and to a lesser extent, pseudoazurin and eukaryotic plastocyanins. Id. Hydrophobic patches are also found to a lesser extent in steliacyanin and rusticyanin copper sites, but have different features. Id.

Table 1. Sequence and structure alignment of azurin (1 JZG) from P. aeruginosa to other proteins using VAST algorithm.

PUB Alignment % aa P-ralue Score RMSD Description Ien th' identi IAOZ A 2 82 18.3 10 c-7 12.2 1.9 Ascorbate oxidase 1QHQ A 113 31 1 Oe-7.4 12.1 1.9 AuracyaninB
lV54 B 1 79 20.3 10e-6.0 11.2 2.1 Gytocrome c oxidase 1 GY2 A 9~) 163 10e-5.0 11.1 1.8 Rusticyanin 3~}VISI' A 74 8.1 l De-6.7 10.9 2.5 1Vlotile Major Sperm Prestein5 74 G~.z loe-5.6 10.3 2.3 plastoc.va.nin lKt.~Y E 90 5.6 1C3e-4.6 lO.l 3.4 Ephrir,L,' IPMY 17.I iOc:-4.1 9.8 2.3 F'seaid(%_j.~.Mn ?0 r Ali~med Length: The number of equivalent pairs of C-alpha atorns superimposed between the two stru.ctures, i.e. how many residues have been used to calculate the 3D
superposition.

..,r., A.L: Tla, ti . ST p _! ~c a ze:,,, ,- 11~,: s; c:e of tb~, c, A,.I:
~
.~5 -I9..

against seeing a match of this quality by pure chance. The p value from VAST
is adjusted for the effects of multiple comparisons using the assumption that there are 500 independent and unrelated types of domains in the iVMDB database. The p value shokvn thus corresponds to the p value for the painvise comparison of each domain pair, divided by 500.
3 Scarc: The VAST structure-sirriilarity score. This number is related to the n.umber of secondary structure elements superimposed and the quality of that superposition. Higher VAST scores correlate with higlier similarity.
4 RNISD: The root mean square superposition residual in Angstroms. This number is calculated after optimal superposition of two structures, as the square root of the mean square distances between equivalent C-alpha atoms. Note that the RMSD value scales with the extent of the structural aligmments and that this size must be taken into consideration when using RMSD as a descriptor of overall structural similarity.
C elegans major sperm protein proved to be an ephrin antagonist in oocvte maturation. Kuwabara, Genes and Development I 7: I S5- I 61 (2003), Azurin The azurins are copper containing proteins of 128 amino acid residues which belong to the family of cupredoxins involved in electron transfer in certain bacteria. The azurins include those from P. aeruginosa (PA) (SEQ ID NO: I), A. xylosoxidans, and A.
denitrificaris. Murphy et al., J. Mol. Biol. 315:859W871 (2002). The amino acid sequence identity between the azurins varies between 60-90 /a, these proteins showed a strong structural homology. All azurins have a characteristic 0-sand3,vich with Greek key motif and the single copper atom is always placed at the sarne region of the protein. In addition, azurins possess an essentially neutral hydrophobic patch surrounding the copper site.
Id.
?5 Plastocyanins rne: plastocyanins are soluble proteins of cyanr.~b;~ c7 ,i, algae <md plants that contain one molecule of copper per molecule a-nd are blue i~-, t1h~ oxiciired form.
Tbey occur in the ch.IoroPlast, where they function as electron earriers. Since the detemiiriation of the structure of poplar plastocyanin in 1978, the structure of algal (Scenedesmus, E-tzteromarpha, (.FdCr ,-aonas) and plant (Fz erià b bean~ rIastocya.nins has be.en. dete--mieither by = - -- - , _ . ,' , _ .

laminosikrn, a thermophilic cyanobacterium. Another plastocvanin of interest is from Ulva ~ertzrssis.

Despite the sequence divergence, among plastocyanins of algae and vascular plants (e.g., 62% sequence identitv between the Chlamydomonas and poplar proteins), the three-dimensional structures are conserved (e.g., 0.76 A rms deviation in the C
alpha positions between the C1alamvclomonas and Poplar proteins). Structural features include a distorted tetrahedral copper binding site at one end of an eight-stranded antiparallel beta-barrel, a pronounced negative patch, and a flat hydrophobic surface. The copper site is optimized for its electron transfer function, and the negative and hydrophobic patches are proposed to be involved in recognition of physiological reaction partners. Chemical modification, cross-linking, and site-directed mutagenesis experiments have confirmed the importance of the ne,ative and hvdrophpbic patches in binding interactions with cytochrome f, and validated the model of two functionally signifcant electron transfer paths involving plastocyanin. One putative electron transfer path is relatively short (approximately 4 A) and involves the solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other is more lengthy (approximately 12-15 A) and involves the near3v conserved residue Tyr-83 in the negative patch. Redinbo cr al., J. Bioenerg. Biomembr. 26:49-66 (1994).

Rusticyanins Rusticyanins are blue-copper containing single-chain polypeptides obtained from a Thiobacillus (now called Acidithtobacillus). The X-ray crystal structure of the oxidized form of the extremely stable and highly oxidizing cupredoxin rusticyanin from Thiobacillus ferrovxiclans (SEQ ID NO: 4) has been determined by multiwavelength ation-ialous diffraction and refined to I.9A resolution. The rusticyanins are composed of a core beta-sandwich fold composed of a six- and a seven-stranded b-shett_ Like other cupredoxilis. the copper ion is coordinated by a cluster of four eonscrved (His 85, Cysl38. I-Iis143, Metl48) arranaed i.n a distorted tetrahedron. Walter, R.L. ei a.l.r J. Mol.
Biol. 263: 730-51 (1996).

Pseudoazurins The p-c.udoarurins are a far-ntly of blue-c s3pl,vr containir ~~ngle-chain polypeptide.
~ õ_ , = , ; _ ~ - , . .

similar structure to the azurins although there is low sequence homology between these proteins. Two rnain differences exist between the overall structure of the pseudoazurins and azurins. There is a carboxy terminus extension in the pseudoazurins, relative to the azurins, consisting of two alpha-belices. In the mid-peptide region azurins contain an extended loop, shortened in the pseudoazurins, which forms a flap containing a short a-helix.
The only major differences at the copper atom site are the conformation ot`the MET side-chain and the Met-S copper bond length, which is significantly shorter in pseudoa2,urin than in azurin.
Phytocyanins The proteins identifiable as phytocyanins include, but are not limited to, cucumber basic protein, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative blue copper protein in pea pods, and a blue copper protein from Arabidopsis tliccliana. In all except cucumber basic protein and the pea-pod protein, the axial methionine ligand normally found at blue copper sites is replaced by glutamine.
Auracyanin Three small blue copper proteins designated auracyanin. A, auracyanin B-1, and auracyanin B-2 have been isolated from the thermophilic green gliding photosynthetic bacterium Chloroflexus atirQntiaczis. The two B forms are glycoproteins and have almost identical properties to each other, but are distinct from the A form. The sodium dodecyl sulfate-polyacryIamide gel electrophoresis demonstrates apparent monomer molecular masses as 14 (A), 18 (B-2), and 22 (B-I) kDa.
The amino acid sequence of auracyanin A has been determined and showed auracyanin A to be a polypeptide of 1.39 residues. Van Dreissche et al., Protein Science 8:947-957 (1999). His58, Cysl'3, His128, and Met132 are spaced in a way to be expected if they are the evolutionary conserved metal ligands =:Ts in known small copper proteins plastocyanin and azurin. Secondary structure predicticr also indicates that a-~-acyanin has a general beta-barrel structure similar to that of azurin. from Pseudolnoncas aeruginosa and plastocyanin from poplar leaves. However, auracyanin appears to have sequence characteristics of both small copper protein sequence classes. The overall similarity with a ii _: c ~. _ .orice ot _7'1-remaxkably rich in glycine and hydroxy amino acids. Id. See exemplary amino acid sequence SEQ ID NO: 15 for chain A of auracyanin from Ghluraflexus aurantiaeus (NCBI
Protein Data Bank Accession No. A.A~'Ll 12874).
The auracyanin B molecule has a standard cupredoxin fold. The crystal structure of auracyanin B from CliI roflexus aurantiacus has been studied. Bond et al., J.
Mo1, Biol.
306:47-67 (2001), With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurin. As in other cupredoxins. one of the Cu ligands lies on strand 4 of the polypeptide, and the other three lie along a large loop between strands 7 and 8. The Cu site geometry is discussed with reference to the a-Tnino acid spacing between the latter three ligands. The crystallographically characterized Cu-binding domain of auracyanin B is probably tethered to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits significant sequence identity with known tethers in several other membrane-associated electron-transfer proteins. The amino acid sequences of the B forms are presented in Mc?1!Ianus et al. J. Biol. Chem. 267:6531-6540 (1992). See exemplary amino acid sequence SEQ ID NO: 16 for chain B of auracyanin from Chioraflexus aurantiaciss (NCBI Protein Data Bank Accession No. 1QHQA).

Stellacyanin Stellacyanins are a subclass of phytocyanins, a ubiquitous family of plant cupredoxins. An exemplary sequence of a stellacyanin is included herein as SEQ
ID NO: 14.
The crystal structure of umecyanin, a stellacyanin from horseradish root (Koch et aL, J. Am.
Chem. Soc. 127:158-166 (2005)) and cucumber stellacyanin (Hart el al., Protein Science 5:2175-2183 (1996)) is also known. The protein has an overall fold similar to the other phytocyanzns. The epllrix B~', protein ectodomain tertiary structure bears a significant similarity to stellacyanin. Toth et al., Developmental Cell 1:83-92 (2001). An :_,xemplary arnino acid sequence of a stellacyanin is found in the Center for i1iu t~~_Ir,ologyy lnforTnation Protein Data Bank as Accession No. 1JER, Sh- Q ID NO: 14.

Cucumber basic protein ~(1 An exemplary arnilio acid sequence from a cucumber basic protein is included herein as SEQ ID NO: l~. The cr= s fa1 ~fructnre of t~ry c. t: rhc, b._.<;c: pr;-=tr~i~', (CBP), d i.~ I blue . , , ',. .. ._ :. .. '.. _~.. _ _ .,. . _ ---~

one side and is better desc>-ibed as a` beta-sandw icb" or "beta-taco". Guss ct al., J. Mol. Biol.
262:686-705 (1996). The ephrinB2 protein ectodomian tertiarv structure bears a high similarity (rms deviation 1.5A for the 50 cz carbons) to the cucumber basic protein. Toth et al., Developmental Cell 1:83-92 (2001).
The Cu atom has the norz-nal blue copper NNSS' co-ordination with bond lengths Cu-N(His39) = 1.93 A, Cu-S(Cvs79) =?.16 A, Cu-N(His84) = 1.95 A, Cu-S(MetS9) =~.61 A.
A disulphide link, (Cys52)-S-S-{Cy-s85). appears to play an important role in stabilizing the molecular struct-ure. The polypeptide fold is typical of a sub-family of blue copper proteins (phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3, with which CBP has a high degree of sequence identity. The proteins currently identifiable as phytocyanins are CBP, stellacyanin, mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative blue copper protein in pea pods, and a blue copper protein from Arabidopsis thaliana. In all except CBP and the pea-pod protein, the axial methionine ligand norinally found at blue copper sites is replaced by glutamine. An exemplary sequence for cucumber basic protein is found in NCBI Protein Data Bank Accession No. 2CBP, SEQ ID NO: 17.
Nlethods of Use The invention provides methods to prevent de novo malignancies in otherwise healthy patients comprising administering to the patient at least one peptide that is a cupredoxin, or variant, derivative or structural equivalent thereof, as described above.
Chemopreventive therapies are based on the hypothesis that the interruption of processes involved in cancergenesis will prevent the development of cancer. The cupredoxin Psetcdornonas aeniginosa azurin and the truncated azurin peptide p28 are now known to inhibit the development of premalignant lesions, either bv inhibiting the initial fomlation of 2 5 premalignant lesions, or killing or inhibiting the growth of premaIignant lesions that are present. It therefore contemplated that a cupredoxin, or variant, derivative or structural equivalent thereof, as described above, with the L-bility to inhibit the development of premalipazat lesions, may be used in chemc,prf:-, c., =ive therapies in otherwise he:alth}, patients. Such othemrise healthy patients are, in ,,ome e-mbodirrrents.
patients at a higher risk to develop cancer than those in the general population. Cancers tb.at may be prevented b;r 1 1t ~,t E
tiI \~
S ,Y^^'A:'..JY ll ':\ i,) :~ I : C_~~ - t. - 1~~~ ~ 1, ..~t .L1 { i= I.~Y 1 .. .a ..._ ._,.. - _ . . I:.,~~._.

prostate, skin, and cervical cancer. In some embodiments, the patient may be human. In other embodiments, the patient is not human.
The invention further includes methods to study the development of cancer comprising contacting mammalian cells before or after induction with a carcinogen with a composition carrtprisirlg. cupredoxin, or a vartant, derivative or structural equivalent thereof and observing the development of the cells. In some embodiments, the cells are mouse mammary gland cells, while in others they are other cells that may become malignant in mammals.
Patients at a higher at risk to develop cancer than the general population may be patients with high risk features, patients with premalignant lesions, and patients that have been cured of their initial cancer or definitively treated for their premalignant lesions. See generally Tsao et al., CA Cancer J Clin 54:150-180 (2004). High risk features may be behavioral, genetic, environmental or physiological factors of the patient.
Behavioral factors that predispose a patient to various forms of cancer include, but are not limited to, smoking, diet, alcohol consumption, horznone replacement therapy, higher body mass index, nulliparity, betal nut use, frequent mouthwash use, exposure to human papillomavirus, childhood and chronic sun exposure, early age of first intercourse, multiple sexual partners, and oral contraceptive use. Genetic factors that predispose a patient to various forms of cancer include, but are not limited to, a family history of cancer, gene carrier status of B.RCA.1 and BRC<42, prior history afbreast neoplasia, familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (HNPCC), red or blond hair and fair-skinned phenotype, xeroderma pigmentosuni, and ethnicity. Environmental features that predispose a patient to various forms of cancer include, but are not limited to, exposure to radon, polycvclic aromatic hy=drocarbons, nickel, chromate, arsenic, asbestos, chloromethyl etlh~ rs.

_2 _5 benzo[a?p)~Tene, radiation, and aromatic amines from .'uK,~.- - pa.irit occupational Other miscellaneous factors that predispose a patient to ~arious forms of ~~ancer include, but are not limited to, chrcan.ic, obstrat... i ~;i: m,,rv disease cvi.th airflow c on, chronic bladder infections. schistosom: i ;s, older a,:;e, and immunocompromised stutus.
Additionally, patients at. a higher risk of developing cancer may be detez-mined by the use ofvariot;s ri>k: mot;L:1,. that beÃ,- d~L-f~~pi~d for cQ~ttairi kin.is ot cancer. For orthe Claus model, araong others. See Gail et aL, JNatl Cancer Inst 81:I879-1886 (1989);
Cuzick, Breast 12:405-411 (2003); Huang er czl., Am J Epidemiol 151:7034714 (2000).
Patients with premalit-.nant lesions are at a higher risk to develop cancer than the general population. T'he presence of premalignant lesions in or on a patient may be determined by many methods that are well known to those in the art.
Intermediate markers or biomarkers that originate from premalignant lesions may be measured in a patient to determine if the patient harbors prerrzaligmant lesions. Chromosomal abnormalities occur in tumor cells and the adjacent histologicially normal tissues in the majority of cancer patients.
Progression in chromosonnal abnormalities parallels the phenotypic progression from premaligna-nt lesion to invasive cancer. Thiberville et al., Cancer Res.
55:5133-5139 (1995).
Therefore, chromosomal abnorrnalities associated with cancer may be used as intermediate markers to detect premalignant lesions in a patient. Common chromosomal abnormalities associated with cancer include, but are not limited to, allelic deletions or loss of heterozygosity (LOH) in tumor suppressor genes such as 3p (FHIT and others), 9p (9p21 for 1 Er~x415and .~ g"~~zr p , p , p ), 17p (17p13 for p53 gene and others) and 13q (13q14 for retinoblastoma gene Rb and others). Deletions in 3p and 9p are associated with smoking and the early stages oflun.g cancer. Mao et al., J. ~Natl. Cancer Inst. 89:857-862 (1997).
Deletions affecting 3p, Sq, 8p, 17p and 18q are common change in epithelial cancers. See gcncrally Tsao et al.. CA Clin. Cancer J. Clin. 54:153 (2004). Other chromosomal mutations associated with cancer include those which activate oncogenes. Oncogenes whose presence may be used as intermediate markers include, but are not limited to, Ras, c-myc, epidemral growth factor, erb-B2 and cyclins E, D,t and.BI. See generally id. at 154.
Other intermediate markers may be the products of genes up-regulated in premalignant cells and cancer cells. Genes that may be up-regulated in premalignant cells include:, but are not Iirnited to, cyc1oc~xygrc:lcCOX-1 an.d COX-2, telomerase. Other biornarkers of cancer cells, and sorne premaii~wa,nt cells, incIade. but are not limited tcx, p_53~
e-pidennal growth factor receptor (GFR), proliferating cell nuclear ~l'CNAj, RAS.
COX-2, Ki-67, DNA aneuploidy, DNA polymerasc-a, ER. Her2neu, L,aclberin, RARO, hTERT, pM` F
I--IIT (3p14), Bcl-2, VEGF-R, HPV infection, LOH 9p'.I, LOH l'p, p-VKT, hnRNP A2/f31, RAF, Myc, c-KIT, c-, ~-Iin L) l ,E and B 1. ICsp' t_ bel-2, ~.~1 6, LOH
~. 3, LOH 3~~2'~_ LOH 9P LOH I. LOH :l'C DCC, -OPC4; JVI 8, BAX, pSA, u` ` M i , N:F-kb, Air ' FPV . _ l k I _Oi LOH 5p, bladder tumor antigen (BTA), BTK TRAK (Alidex, Inc., Redmond WA), urinary tract matrix protein 22, fibrin degradation product, autodrine motility factor receptor, BCLA-4, cvtokeratin 20. hygaluronic: acid, CYFRA 2I-1, BCA, beta-human chorionic gonadotrapin, and tissue polypeptide antigen (TPA). See gerzerally, icl, at 1 S5-t5 7.
Patients that have been cured of their initial cancers or have been definitively treated for their prernalignant lesions are also at a higher risk to develop cancer than the aene,ral population. A second primarv tumor refers to a new primary cancer in a person with a history of cancer. Second primary tumors are the leading cause of mortality-in head and neck cancer. Id. at 150. A second primary tumor is distinct from a metastasis in that the former originates de novo while the later originates from an exi,ting tumor. Patients that have been cured of cancer or premalignant lesions of the breast, head and neck, lung, and skin are at a particularly high risk to develop second primary tumors.
The compositions comprising a cupredoxin or variant, derivative or structural equivalent thereof can be administered to the patient by many routes and in many regimens that will be well know-n to those in the art. In specific embodiments, the cupredoxin, or variant, derivative or structural equivalent thereof is administered intravenously, intramuscularly, subcutaneously, topically, orally, or by inhalation. The compositions may be administered to the patient by any means that delivers the peptides to the site in the patient that is at risk of developing cancer. In specific embodiments, the cupredoxin or variant, derivative or structural equivalent thereof is administered intraveneously.
In one embodiment, the methods may comprise co-administering to a patient one unit dose of a composition comprising a cupredoxin or a variant, derivative or structural equivalent of cupredoxin and one unit dose of a composition comprising another chemopreventive drug, in either order, administered at about the same time, or within about a given time following the adrninistration of the crth~,~r. for example. about one minute to about 6o minutes following the adrn,f rrf stra.tion of the other drue, or about 1 hour to about 12 hours following the administration of the other drug. Chemopreventive drugs of interest include, but are not limited to, tarriox.ifen. aromatase infiibitors such as letrozole and anastrozole ~A.rimidexretinoids such as N-[4-hvdroxyphenyll retinamide (4-HPR, fenretinide), nonsteriodal antiinfla.r-jnr to~-y ~~zen: (V-AID:~) ~uciz as _:,irin an.l ~,ulindac, celecoxib ~t (COX G II2h1bItor cid <~ ~ s F I.-L
GfR ,,: -i: .
..Z7 -(antibody tc) VEGF-receptor}, cetuximab (antibody to EGFR), retinol such as vitamin A, beta-carotene, I3-cis retinoic acid, isotretinoin and retinyl palmitate, rt-tocopherol, interferon, oncolytic adenovirus dI 1520 (0NYX-(I15), ;efitinib, etretinate, finasteride, indole-3-carbinol, resveratrol, chlorogenic acid, raloxiferie, and oltipraz.

4; Pharmaceutical Compositions Comprising Cupredoxin, Or Variunt, Derivative Or Structural Equivalent Thereof Pharmaceutical compositions comprising cupredoxin or variant, derivative or structural equivalents thereof, can be manufactured in any conventional manner, e.g., by conventional mixing, dissolviug, granulatitig, dragee-making, emulsif~jing, encapsulating, entrapping, or lyophilizing processes. The substa.ntially pure or pharmaceutical grade cupredoxin or variants, derivatives and structural equivalents thereof can be readily combined with a pliarmaceutically acceptable carr-ier well-known in the art. Stich carriers enable the preparation to be formulated as a tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, and the like. Suitable carriers or excipients can also include, for example, fillers and cellulose preparations. Otber exeipients can include, for example, flavoring agents, coloring agents, detackifiers, thickeners, and other acceptable additives, adjuvants, or binders.
In some embodiments, the pharmaceutical preparation is substantially free of preservatives.
In other embodiments, the pharmaceutical preparation may contain at least one preservative.
General methodology on pharmaceutical dosage forms is found in Ansel et al., Pharmaceutical Dosage F rns and Drug Delivery Systems (Lippencott Williams &
Wilkins, Baltimore MD ( I 999)).
The composition comprising a cupredoxin or variant, derivative or structural equivalent thereof used in the invention may be administered in a variety of ways, including by injection (e.g, intradermal, subcutaneous, intramuscular, intraperitoneal aizci the like), by inhalation, by topg.;~' administrati.ol:i, by suppository, I) ,, atransclermai patch or bv mouth. Generai ir.!, rin._tiofl or U'.~,~ dc';very systems can be i`ouncl in Ao-,-,c' ial., id.. In some ernbod.iment~a,, he c~.~srrspositic=,i comprising a cupredoxiii or va.riant, u!crivative or structural equivalent thereof can be fort-rmulated and used directly as injee:tibles. for subcutaneous and intravenous injection, among others. The injectable frsrrrmulation, in a~~ particular, can acivanta~eoaslv be~ used to tr~;at. patier~tq thrt aI?p-c~priate i~~r ;~; , , ~_=__ _ _ , structural equivalent thereof can also be taken orally after mixing wit1a protective agents such as polvprespylene g.lvcols or similar coating agents.
Whezi administration is by injection, the cupredoxin or variant, derivative or structrzral Gcluivalent thereof may be formulated in aqueous solutions, specifically in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer.
The solution may contain formulatorv agents such as suspending, stabilizing and=Or dispersing agcnts. Alternatiti-ely, the cupredoxin or variant, derivative or structural equivalent thereof mav be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-f.ree water, before use. In some embodiments, the pharmaceutical cornposition does not I0 comprise an adjuvant or any other substance added to enhance the immune response stimulated by the peptide. In some embodiments, the pharmaceutical composition comprises a substance that inhibits an immune response to the peptide.
When administration is by intravenous fluids, the intravenous fluids for use administering the cupredoxin or variant, derii ative or structural equivalent thereof may be composed of crystalloids or colloids. Crystalloids as used herein are aqueous solutions of mineral salts or other water-soluble molecules. Colloids as used herein contain larger insoluble molecules, such as gelatin. Intravenous fluids may be sterile.
Crystalloid fluids that may be used for intravenous administration include but are not limited to, normal saline (a solution of sndium chloride at 0.9 io concentration), Ringer's lactate or Ringer's solution, and a solution of 5% dextrose in water sometimes called D5W, as described in Table 2.

Table 2. Composition of Common Crystalloid Solutions Solution Other Name [Na_~] [Cf-I iGlucasel D5W 5% Dextrose 0 0 215 2 2f3 & I'.3 3. 3% Dextrose :5 1 - I 168 0.~v~ saline Half-normal 0.45% .hiaCl 77 77 0 saline Normal saline W)" :J Na.CI I _5 4 154 0 130 l.0~-~ ~i *Ringer's lactate also has 28 mmol/L lactate, 4 mmoliL K# ajid 3 mmoli'L Ca~~.

When administration is by inhalation, the cupredoxin or variant, derivative or structural equivalent thereof may be delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.~., dichlorodifluoromethane, trichlorotluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be detÃ:rmineci by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., belatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the proteins and a suitable powder base such as lactose or starch.
When administration is by topical administration, the cupredoxin or variant, derivative or structural equivalent thereof niay be formulated as solutions, gels, ointments, creams, jellies, suspensions, and the l'zke, as are well known in the art. In some embodiments, administration is by means of a transdermal patch. When adrriinistration is by suppository (e.g., rectal or vaginal), cupredoxin or variants and derivatives thereof compositions may also be formulated in compositions containing conventiozial suppository bases.
When administration is oral, a cupredoxin or variant, derivative or structural equivalent thereof can be readily formulated by combining the cupredoxin or variant, derivative or structural equivalent thereof with pharmaceutically acceptable carriers well known in the art. A solid carrier, such as mannitol, lactose, magnesium stearate, and the like may be employed; such carriers enable the cupredoxin and variants. derivatives or structural equivalent thereof to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, cellulose preparation, granulatizl;:; z-ents, and binding agents.
Other convenient carriers, as wcll-knowzi in `Ii:_ <.Iso inelude multivaterit carriers, such as bacterial capsular polvsaccharide, a dex.tranor _ L,eneticallyerr(;ineered vector. In addition, sustained-release formulations that include a cupredoxin or variant, derivative or structural eclun <c,v thereof allow for the release of cupredoxin or variant, derivative or structural equival~nt therecrfover extended periods of time, such tbat without the suo::ined rL1~./4L.SV. l~-Jl.~:il--`~AtZon.y the variant, dY:4Ik'ak-t~'4= ~.: _ .. o Li.&I"-iilk.' LCIF l.._ii=

would be cleared from a subject's system, artd~Or degraded by, for example, proteases and simple h~;-drolysis before eliciting or enltancing a therapeutic effect.
The half-life in the bloodstream of the peptides of the invention can be extended or optimized by several methods well known to those, in the art. The peptide variants of the inr:-ention may include, but are not limited to, various variants that may increase their stabilitv, specific activitv, Iongevitv in the bloodstream, and,t`or decrease immunogenicity of the cupredoxin, while retaining the ability of the peptide to inhibit the development of premalignant lesions in mammalian cells, tissues and animals. Such variants include, but are not limited to, those which decrease the hydrolysis of the peptide, decrease the deamidation of the peptide, decrease the oxidation, decrease the immunogenicity, increase the structural stability of the peptide or increase the size of the peptide. Such peptides also include circularized peptides (seeMotzk et al., Bi Drcxgs 19(4):261-78, (2005);
DeFreest et al., J. Pept. Res. 63(5):409-19 (2004)), D,L-peptides (diastereomer), Futaki et al., J. Biol. Chem. Feb 23;276(8):5836-40 (2001); Papo et al., Cancer Res. 64(1 b):5779-86 (2004); Miller et al., Biochem. Pharmacol. 36(1):169-7E, (1987)); peptides containing unusual amino acids (see Lee et al., J. Pept. Res. 63(2):69-84 (2004)), N- and C- tertn.inal modifications (see Labrie et al., Clin. Invest. Med. 13(5):275-8, (1990)), hydrocarbon stapling (see Schafineister et ccl., J. Am. Chem. Soc. 122:5891-5892 (2000);
Walenski et al., Science 305:1466-1470 (2004)} and PEGylation.
In various embodiments, the pharmaceutical composition includes carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents and/or preservatives), water, oils, saline solutions, aqueous dextrose and glvcerol solutions, other pharmaceutically acceptable auxiliary 15 substances as recluire;d to approximate ph;vi;, L;~al conditj.,.;,_ uch as bu_-; o_~Lnts, tonicity adjusting agents, wetting agents arid t1-ie like. It wili e,:
recognized tI~ .:. ixhile artv suitable carrier known to those of ordittarw skill in the aft may be employed to a.clmillistcr the compositions of this invention, the tv _ pe of carrier will vary depending on the mode of administration. Compounds may also be encapsulated within liposomes using well-known 3I1 te~ I~~c~Ir,gv. Bic$def,--adable zn.icrosphereti rrti,~N'v al~r, he e~.~plox; cXr1;a~ clr~rferiz tor the -3~-disclosed, for example, in U.S. Patent N-5os. =1,89'11,268; 5,075,109;
5,928,6=17; 3,811.I28;
5,820,883; 5,85-1,763; S,814,344 and 5,942,252.
The pharmaceutical compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. 1 he resulting aqueous solutions may be packaged for use as is, or tyopbilized, the lyophilized preparation being combined with a sterile solution prior to admiziistration.

Administration of Cupredoxin or Variant, Derivative or Structural Equivalent Thereof The cupredoxin or variant, derivative or structural equivalent thereof can be administered formulated as pharmaceutical compositions and administered by any suitable route, for example, by oral, buecal, inhalation, sublingual, rectal, vaginal, transurethral, nasal, topical, percutaneous, i.e., transdermal or parenteral (including intravenous, intramuscula.r, subcutaneous a.iid intracoronary) or vitreous administration. The pharmaceutical formulatioiis thereof can be administered in any amount effective to achieve its intended purpose. More specifically, the composition is administered in a therapeutically effective amount. In specific embodiments, the therapeutically effective amount is generally from about 0.0 1-20 mg/day/kg of body weight.
The compounds comprising cupredoxin or variant, derivative or structural equivalent thereof are useful for the prevention of cancer, alone or in combination with other active agents. The appropriate dosage will, of course, vary depending upon, for example, the compound of cupredoxin or variant, derivative or structural equivalent thereof employed, the host, the mode of administration and the nature and severity of the potential cancer.
However, in general, satisfactory results in liumans are indicated to be obtained at daily dosages from about 0.01-20 mg'kg of body weight. An indicated daily dosage in humans is 2_5 in the range from about 0.71 mg to about 1400 m,; of a compound of cupredoxin or variant, des v.ative or sta: a~aural equivalent thereof cc~~ ~~Jicrttly for example, in daily do;c~ ,: eelCl monthly doses, and,-or continuous d-i_ig. Du.ily doses e.an bein discrete dosages from I to 12 times per day. Altematively, doses can be adiniiiistered every other day, every third day, every fourth day, every fifth day, every sixth day, every week, and sin-iilarly in day increments up to 31 days or over. Alternatively, dosing can be continuous using patcbe4. i_v. administratio i~.; i.'id- like.

_ __.- ------ -- - - -- -__~~.
The exact formulation, route of administration, and dosage is determined by the attending physician in view of the patient's eonditi.on. Dosa~e amount and interval can be adjusted individually to provide plasma levels of the active cupredoxin or variant, derivative or structural equivalent thereof which are sufficient to maintain therapeutic effect. Generally, the desired cupredoxin or variant, derivative or structural equivalent thereof is administered in an admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharrnaceutical practice.
In one aspect, the cupredoxin or variant, derivative or structural equivalent thereof is delivered as DNA such that the poiypcptide is generated in situ. In one embodiment, the DNA is "naked," as described, for exarnple, in Ulmer et al., (Science 259:1745-1749 (1993}j and reviewed by Cohen (Science 259:1691-1692 (1993)). The uptake of naked DNA
may be increased by coating the DNA onto a carrier, e.g., biodegradable beads, which are then efficiently transported into the cells. In such methods, the DNA may be present within any of a varietv of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. See, e.g., W090/11092,'V4093/24640, WO 931-17706, and U.S. Pat. No.
5,736,524.
Vectors, used to shuttle genetic material from organism to organism, can be divided into two general classes: Cloning vectors are replicatizig plasmid or phage with regions that are essential for propagation in an appropriate host cell and into which foreign DNA can be inserted; the foreign DNA is replicated and propagated as if it were a component of the vector. An expression vector (such as a plasmid, yeast, or animal virus j~enome) is used to introduce foreign genetic material into a host cell or tissue in order to transcribe and translate the foreig.n DNA, such as the DNA of a cupredoxin, T.n expression vectors, the introduced DNA is operably-linked tc) elements stzch as promoters that sign.ii :;~ ccll to highly tr~u-iscri~e tb.c inserted DNA. Some promoters are exceptionally us_=t;r s;_,,_:?i as inducible pro,~v.,-s that control gene transcription in io specific factc?r:-.
Opera.blvrlinlCinga cupredoxin and variants and derivatives thereof pcil~nucleotide to an inducible promoter can control the expression of the crzprcdoxin and variants and derivatives thereof in response to spe~ i tic fac o ,. Exa~iiples of cil ,ic inducii'rle prOT7 Ot~!'~;;-clu&
thosc tl;~ l are resp( nsive to , ihoc~, .. ., . cI~,: nd 5:4,1-ii ~Ãqq$

~_...W. _. ,. : promQtcrs include those that are not endogenous to the cells in which the construct is being introduced, but, are responsive in those cells when the induction agent is exogenously supplied. In gerleral. useful expression vectors are often pla;rnids. However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) are contemplated.
Vector choice is dictated by the organism or cells being used and the desired fate of the vector. In general, vectors eomprise signal sequences, origins of replication, marker genes, polylinker sites, enhancer elements, promoters, and transcription termination sequences.
Kits Comprising Cupredoxin, or Variant, Derivative Or Structural Equivalent Thereof In one aspect, the invention provides regimens or kits comprising one or more of the following in a package or container: (1) a pharmacolQgically active composition comprising znl at least one cupredoxin or variant, derivative or structural equivalent thereof; (2) an additional chemopreventive drug, (3) apparatus to administer the biologically active composition to the patient, such as a syringe, ne:bulizer etc..
WThen a kit is supplied, the different components of the composition may be packaged in separate containers, if appropriate, and admixed immediately before use.
Such packaging of the components separately may perrrmit long-term storage without losing the active components' fiznctions.
The reagents included in the kits can be supplied in containers of any sort such that the life of the different components are presenred and are not adsorbed or altered by the inateria.ls of the container. For example, sealed glass armpules may contain lyophilized cupredoxin and variants, derivatives and structural equivalents thereof, or buffers that have 2 5 been packaged under a neutral, non-reacting gas, such as nitr~.~gcn..
Arzlsules may consist of any suitable material, such as glass, organic polyrners, such as poiyc:arbonate, polystyrene, etc., ceramic, ix-iet.al or any other material typically eniployed to b.old similar reagents. Other examples of suitable containers include simple bottles that may be fabricated from si.rrrilar substances as ampules, and envelopes, that may comprise foil-lined interiors, such as 3{) aluminum or an alloy. Other con1nei~ i_iJude test tubu:,, vials, tla1 sks, bottles, syringes, or hottle havi that can &x~ pi~r~ccib ~a tS

that are separated by a readily removable me7nbrane that upon removal permits the components to be mixed. Rernovable membranes may be glass, plastic, rubber, etc.
Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and-or may be supplied as an electronic-readable mediurn, such as a floppy disc. CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, tlasb memory device etc.
Detailed instructions may not be physically associated with the kit; instead, a user rnay be directed to an intemet web site specified by the manufacturer or distributar of the kit, or supplied as electronic mail.

Modification of Cupredoxin and Variants, Derivatives and Structural Equivalents Thereof Cupredoxin or variant, derivative or structural equivalents thereof may be chemically modified or genetically altered to produce variants and derivatives as explained above. Such variants and derivatives may be synthesized by standard techniques.
In addition to naturally-occurring allelic variants of cupredoxin, changes can be introduced by mutation into cupredoxin coding sequence that incur alterations in the amino acid sequences of the encoded cupredoxin that do not significantly alter the ability of cupredoxin to inhibit the development of premalignant lesions. A "non-essential" amino acid residue is a residue that can be altered fr tn the wild-type sequences of the cupredoxin without altering pharmacologic activity, whereas an "essential" amino acid residue is required for such pharmacologic activity. For example, amino acid residues that are conserved among the cupredoxins are predicted to be particularly non-amenable to alteration, and thus "essential."
Amino acids for which conservative substitutions thGa do not change the pb.artnac~.rlogie activity oi'tlie pi 1N 1%pti;Ae can be made ar. , l~ne~~~;n in the a.r:. C,;t,' conservative substitutions are sh.ouaL in Table 3, "Preferred substitutio s."
CConservative substitutions whereby an amino acid of one class is replaced with of the same type fall within the scope of the invention so long as the substitation d{-,cti not materially alter the pharmacologic activity of the compound.

Table 3. Preferred substitutions Original residue Exemplary substitutions Preferred substitutions Ala (A) Val, Leu, Ile Val .Vg ~R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser GIn (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg ile (1) Leu, Val, Met, Ala, Phe, Leu Norieucine Leu (L) Norleucine, Ile, Val, Met, Ala, Ile Pire :Lys (K) Arg, Gln, Asn Arg met (M) Leu, Phe, Ilc Leu Phe (F) Leu, Val, Ilc, Ala, Tyr Leu Pro (P) Ala Ala Ser(S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tvr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Leu Norleucine Non-conservative substitutions that affect (1) the structure of the polypeptide backbone, such as aP-sheet or a-helical conformation, (2) the charge, (3) b.ydrophobicity, or (4) the bulk of the side chain of the target site can modify the pbarmacologic activity.
Residues are divided into g~rouPs based on common side-chain properties as denoted in Table 4. Non-coiiscrvative substitutions entail exchanging a member of onc, of these classes for a,notb~ r, i;,~,s. Substitutit ns maY be introduced into c on.servativ., ~i;t;Wtion sites or more specifiic~_IIYq into non ; r~'r ~~-d sites.

Table 4. Amino acid classes Class Amino acids hydrophobic Norleucine, Met, Ala, Val, Leu, Ile neutral hydrophilic Cys, Ser, Thr acidic Asp, Glu basic Asn, Gln, His, Lys, A.rg disrupt chain conformation Gly, Pro aromatic Trp, Tyr, Phe The variant polypeptides can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR
mutagenesis. Site-directed mutagenesis (Carter, Biochem J. 237:1-7 (1986);
Zoller and Sm.itb, Methods Enzymol. 154:329-350 (1987)), cassette mutagenesis, restriction selection mutagenesis (Wells et al., Gene 34:315-3 )23 (1985)) or other known techniques can be performed on the cloned DNA to produce the cupredoxin variant DNA.
Known mutations of eupredoxins can also be used to create variant cupredoxin to be used in the methods of the invention. For example, the C I 12D and M44KM64E
mutants of azurin are known to have cytotoxic and growth arresting activity that is different from the native azurin, and such altered activity can be useful in the treatment methods of the present invention.

A more complete understanding of the present invention can be obtained by reference to the following specific Exarnples. The Examples are described solely for purposes of illustration and are not intended to limit the scope of'tbe invention. Changes in forzn and substitution ofef=ul% aItnts are conte,i-nplated as ij,. ~}i-.',.:~nces may suggest or render expedient. Although specific terms have been ernploved berein, such terms are intended in a descriptive sense and not lor pur-pc;G , limitati.ons. Modifications and va.riations of the invention as hereinbefore set forth can be made without departing from. the spirit and scope thereof EXAMPLES
Example 1. Effect of peptide P-28 on DM8A-InducedMan:xmarv lesions in the MMOC
Model Tlie mouse mammalary gland organ culture (MMOC) modei allows evaluating efficacy of potentially chemopreventive agents against development of mammary alveolar lesions (MAL) or mammary ductal lesions (MDL) in response to DMBA. DMBA under appropriate incubation conditions form either MAL or MDL based on the hormonal milieu in the medium. Haw thorne et crl., Pharmaceutical Biology 40: 70-74 (2002); Mehta et al., J.
Natl. Cancer Inst. 93: 1103-1106 (2001). Estrogen and progesterone-treated glands in culture develop ductal lesions whereas aldosterone and hydrocortisone-treated glands form estrogen and pragesterone-independezit alveolar lesions. Mammary glands not exposed to a carcinogen or chemopreventive agent, undergo structural regression in the absence of growth-promoting h.orrnones, whereas treatment with DiMBA for the 24-hr period between days 3 and 4 prevents the regression of struct-ures caused by deprivation of horrnones. It is assumed that this is because the glands have lost normal hormonal responsiveness and now have altered their course of development. Generating mammary adenocarcinoma by transplanting transformed cells into syngeneic mice has proved the premalignant preneoplastic nature of these unrepressed areas.
The thoracic pair of mammary glands was excised aseptically from each Balbic mouse, and the glands were divided into several groups. The effects of p28 were evaluated at 4 different dilutions in the medium. Carcinogen treated glands without the test agent served as a measure to deterrrmine percent incidence in the absencc of a chemopreventive agent. An additional control was included to serve as a positive cc.~t;, >r icr chernoprevention. Azurin was i.ucluded in. the medium at 50 p.g,rml conccntration_ po:- alveolar iesions (MAL) stained ~Aarids were evalu<at41 for the incidence of lesions (glaiicis containing anyf lesions as compared to total ni-imher of glands in a given treatment group). For the ductal lesions (M,DL) similar protoUoi was adapted, however, as indicated below in the methods section the hormonal combiziatioii is di ~-ferent fir al .~,Adu- and duo rrd lesion4. The :,i ands u c:-,~:xed in fon-nalin and then prc~.:.> i;_ 'c l. M :1..

hematoxelene and evaluated under micrQscope. Here the multiplicity of ductal lesions between the control and the treatment groups are compared.
Organ Culture I'racedure. The experimental animals used for the studies were young, virgin BALB/c female mice 3 to 4 weeks of age obtained from Charles River, VV
ilmington.
MA. The mice were treated daily by subcutaneous injec:tions with 1jig esstradiol-17(3 ~ 1 mg progesterone for 9 days. This treatment is a prerequisite inasmuch as animals not pretreated with steroids fail to respond to hormones in vitro. The entire culture procedure is described in detail. Jang et al., Science 275:218-220 (1997); Mehta. Eu. J. Cancer 36:1275-1282 (2000); 1Vfehta et ral., J. Natl. Cancer Inst. 89:212-219 (1997); Mehta et al., J. itiatl. Cancer Inst. 93:11 03-11 C}F (2001).

Briefly, the animals were killed by cervical dislocation, and the thoracic pair of mammary glands were dissected out on silk rafts and incubated for 10 days in serum free Waymouth MB7521I medium (5-glands'5 mlfdish). The medium was supplemented with glutamine, antibiotics (penicillin and streptomycin 100 units; ml medium) and growth -promoting hormones, 5pg insulin (I), 5~ig prolactin (P), 1~ig aidosterone (A) and 1 p,g hydrocortisone (H) per ml of inedi-atn for the protocol to induce mammary alveolar lesions (MAL). For induction of ductal lesions (MDL), the medium contained 5 giznl, 5 g/ml P, 0.001 ~tg/ml estradiol 170 and 1ltg,'mi progesterone (Pg). Mehta et al., J.
ivatl. Cancer Inst.
93:1103-1106 (200I). The carcinogen, DMBA (2 pg`ml) was added to the medium between days 3 and 4. For the present study, DMBA was dissolved in DMSO at a final concentration of 4 mg/ml, and 50 lCg I was added to 100 ml medium resulting in 2pg/ml final concentrations. The control dishes contained DMSO as vehicle.

On day 4, DMBA is removed from the medium by rinsing the glands in fresh medium and transferring them to new dishes containing fresh medium without DMBA.
After 10 davs of incubation, the glands were ffnai,i~,.:ncd for another 1=1 days in the medium containing onlv 1(5 ~tg`ml). During the entire c.ult-ufe period, the, glands were maintained at 37C under 95%
O~, and 5% CO2 environment. The c;bemopreverrtive agent was included in the medium during the first ten days of growth -laresmoting phase. The test peptide p'8 was evaluated at 4 concentrations ranging from 12.5 p.giml to 100 Wznl. Azurin was evaluated at 50 u14/ml in tlie medium. TI,-,: pep: d~, :lissc~lved in -:~=~rile w atc.r and filtered p.
ior to use. Tl,,,;

4p I }, l;' f ~~/ t=i___ ' _. _E. 1 ~i~CCII :.. :-)e'r`,E~it. tl ~-. ~. ._' i- i.. Lka.day).
.>..-I l . ... v1 the {_.~

Results were analyzed by Chi-square analysis and Fisber's Exact Test.
Morphametic Analysis of MAL. For examination of MAL, the glands were stained in alum carmine, and evaluated for the presence of tb.L lt-,iona. The glands were scored for the presence or absence of n-iammarv lesions, severity of lesions per gland, a-nd toxicitv of the agent. The glands stored in xylene were evaluated for the presence or absence, incidence, and severity of mammary lesions for each gland under a dissecting microscope.
Viarnmary glands were scored as positive or negative for mammary lesions, and the percent incidence was determined as a ratio of glands exhibiting lesions and the total number of glands in that group. Dilation of ducts or disintegration of mammary structure because of treatment with chemopreventive agent was considered a toxic effect. The data were subjected to statistical analysis for the incidence to determine the effectiveness of the potential chemopreventive agents.
Figure IA shows a representative photograph of alveolar lesions in a DMBA
treated gland and its comparison with a gland that was treated with DMBA along with a chemopreventive agent. The effects of p28 on the development of alveolar lesion are shown in Figures l B-1 G and summarized in Figure 2. The peptide p28 inhibited MAL
formation by 67% at 25 ~tg/m1 concentration. Increasing concentration further up to 100 p.g/ml did not enhance the efficacy of the peptide. The comparison of the peptide with azurin indicated that p28 was as effective as azurin for MAL development. Azurin at 50 g'ml concentration resulted in a 67% inhibition. Statistical analyses indicated that the effect of p28 was statistically signifeant compared to DMBA control at concentrations greater than 12.5 pg/ml (p<0.0I, Fisher's Exact Test; Chi Square analysis).
Histopathological Evaluation of MDL. ForktDL, the glands were processed for histopathological evaluations. The glands were sectioned longitudinally into 5-micron sections and sta.im-d v_ . r~i ~:.,sin hematoxeline. The lont,itudz'nal section of each gland was divided into several fields and each fi.eld was evaluated for ductal lesions.
Mehta cft al., J.
Natl. Cancer Inst. 93:1103-1I06 (12001). Briefly, the entire gland is evaluated under the scope; smaller glands will have fewer total fields as compared to larger illands. 1`hus, each gland will have variable number of fields. Often the number of sections through the ducts also varies Lr~,ttki~~xozn alavid'o -11 :iid, "1 :'=-i, ~~~~o'ts i~.a thc number from group to &oup- p`iel ', ~2 _k,J~ - [... ;.Irl werà -,-~rnpared ~~EL1.P , i,b@ 4.4LlLxEi.D~.-1 EOS field ~vblAG ~ ,.r~.b XtFt 6 . .. :a. 0 <
_m the hyperplasia or atypia and severely occluded glands. A.n.v field containing any of these histological patterns was considered positive for the lesion. The treatment &roups were compared with the controls for the severity and percent inhibition was calculated.
Figure 3 shows a representative ductal lesion. DMB A induces ductal lesions varying from hvperplasia_ atypia to complete occlusion of the ducts. A ratio of ductal lesionsttotal num.ber of ductal sections was detertnined. Again, 12.5 pg;`rrtl concentration ofp~.8 suppressed only 15% of the MDL formation. However, at 25 ~tgf'ml there was a significant inhibition of the lesions comparable to that observed with 50 ug ml azurin.
The efficacy of p28 at concentrations greater than 12.5 grirnl was statistically significant (p<0.0l, Fishers Exact Test). These results are summarized in Figure 4. Often effects of chemopreN entive agents cazi be differentiated between the TMAL and MDL. For exa.inple tamoxifen inhibited the development of MDL but not 1VIAL. It is interesting to note that azurin and p28 inhibited both estrogen and progesterone-dependent ductal lesions as well as independent alveolar lesions.
100011 This example indicates that both p28 and azurin can prevent the development of precancerous lesions in breast tissue. Thus, p28 and azurin may be used as chemopreventive agents in mammalian patients.

Claims (36)

1. An isolated peptide that is a variant, derivative or structural equivalent of a cupredoxin; and that can inhibit the development of premalignant lesions in mammalian tissue.

2. The isolated peptide of claim 1, wherein the cupredoxin is selected from the group consisting of azurin, pseudoazurin, plastocyanin, rusticyanin, Laz, auracyanin, stellacyanin and cucumber basic protein.

3. The isolated peptide of claim 2, wherein the cupredoxin is azurin.

4. The isolated peptide of claim 1, wherein the cupredoxin is from an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenesfaecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylellafastidiosa and Vibrio parahaemolyticus.

5. The isolated peptide of claim 4, that is from Pseudomonas aeruginosa.

6. The isolated peptide of claim 1, which is part of a peptide selected from the group consisting of SEQ ID NOS: 1, 3-19.

7. The isolated peptide of claim 1, to which a sequence selected from the group consisting of SEQ ID NOS: 1, 3-19 has at least 80% amino acid sequence identity.

8. The isolated peptide of claim 1, which is a truncation of the cupredoxin.

9. The isolated peptide of claim 8, wherein the peptide is more than about 10 residues and not more than about 100 residues.

10. The isolated peptide of claim 8, wherein the peptide comprises a sequence selected from the group consisting of Pseudomonas aeruginosa azurin residues 50-77, Pseudomonas aeruginosa azurin residues 50-67, Pseudomonas aeruginosa azurin residues 36-88, and SEQ ID NOS: 20-24.

11. The isolated peptide of claim 10, wherein the peptide consists of a sequence selected from the group consisting of Pseudomonas aeruginosa azurin residues 50-77, Pseudomonas aeruginosa azurin residues 50-67, Pseudomonas aeruginosa azurin residues 36-88 and SEQ ID NOS: 20-24.

12. The isolated peptide of claim 1, wherein the peptide comprises equivalent residues of a cupredoxin as a region of Pseudomonas aeruginosa azurin selected from the group consisting of residues 50-77, residues 50-67 and residues 36-88.

13. A pharmaceutical composition, comprising at least one cupredoxin or peptide of claim 1 in a pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13 which comprises at least two of the cupredoxins or peptides.

15. The composition of claim 13, wherein the pharmaceutical composition is formulated for intravenous administration.

16. The composition of claim 13, wherein the cupredoxin is from an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylellafastidiosa and Vibrio parahaemolyticus.

17. The composition of claim 16, wherein the cupredoxin is from Pseudomonas aeruginosa.

18. The composition of claim 13, wherein the cupredoxin is selected from the group consisting of SEQ ID NOS: 1, 3-19.

19. A method to treat a mammalian patient, comprising administering to the patient a therapeutically effective amount of the composition of claim 13.

20. The method of claim 19, wherein the patient is human.

21. The method of claim 19, wherein the patient is at a higher risk to develop cancer than the general population.

22. The method of claim 21, wherein the cancer is selected from melanoma, breast, pancreas, glioblastoma, astrocytoma, lung, colorectal, neck and head, bladder, prostate, skin, and cervical cancer.

23. The method of claim 21, wherein the patient has at least one high risk feature.

24. The method of claim 19, wherein the patient has premalignant lesions.

25. The method of claim 19, wherein the patient has been cured of cancer or premalignant lesions.

26. The method of claim 19, wherein the pharmaceutical composition is administered by a mode selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, vitreous injection and oral.

27. The method of claim 24, wherein the mode of administration is by intravenous injection.

28. The method of claim 21, wherein the pharmaceutical composition is co-administered with at least one other chemopreventive drug.

29. The method of claim 26, wherein the pharmaceutical composition is administered at about the same time as another chemopreventive drug.

30. A kit comprising the composition of claim 13 in a vial.

31. The kit of claim 30, wherein the kit is designed for intravenous administration.

32. A method to study the development of cancer comprising contacting the mammalian cells with a cupredoxin or peptide of claim 1; and measuring the development of premalignant and malignant cells.

33. The method of claim 32, wherein the cells are human cells.

34. The method of claim 32, wherein the cells are mammary gland cells.

35. The method of claim 32, wherein the cells are induced to develop cancer.

36. An expression vector, which encodes the peptide of claim 1.