WO1999058097A2 - Utilisation d'agents antiprolactine pour le traitement d'affections proliferantes - Google Patents

Utilisation d'agents antiprolactine pour le traitement d'affections proliferantes Download PDF

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Publication number
WO1999058097A2
WO1999058097A2 PCT/US1999/010545 US9910545W WO9958097A2 WO 1999058097 A2 WO1999058097 A2 WO 1999058097A2 US 9910545 W US9910545 W US 9910545W WO 9958097 A2 WO9958097 A2 WO 9958097A2
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Prior art keywords
hprl
cell
prolactin
cells
prl
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PCT/US1999/010545
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English (en)
Inventor
Wen Y. Chen
Thomas E. Wagner
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Greenville Hospital System
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Priority to AU43086/99A priority Critical patent/AU4308699A/en
Publication of WO1999058097A2 publication Critical patent/WO1999058097A2/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2257—Prolactin

Definitions

  • the present invention relates to methods and compositions for inhibiting
  • compositions of the invention may be used in the treatment of benign as well as
  • Prolactin is a 23-kDa neuroendocrine hormone which is
  • the prolactin receptor (“PRLR”) is a member of the cytokine receptor
  • PRLR is homologous to the receptor for GH ("GHR", also referred to as the somatogen receptor) and both belong to the cytokine receptor superfamily (Kelly et al., 1991,
  • hypophysectomy and bromocriptine administration both directed toward decreasing or
  • tamoxifen a GH analog (octreotide), and a potent anti-prolactin (CV 205-502, a
  • prostate tissue (Aragona and Friesen, 1975, Endocrinol. 97:677-684; Leake et al., 1983,
  • prostate hyperplasia have been reported to be either increased (Odoma et al., 1985,
  • prostate cell lines can be significantly modulated by PRL (1996, Cancer 77:144-149).
  • prostate (Nevalainen et al, 1997, J. Clin. Invest. 99:618-627) may be an important factor.
  • phosphorylated PRL having a bulky negatively charged amino acid (namely glutamate or aspartate) substituted for the serine at position 179 antagonized the growth-promoting
  • hGH human GH
  • binding site 2 (involving the helix 3 glycine of GH mutated in the G120R variant) to
  • the second binding site is sterically hindered and the GH can no longer
  • hPRL human PRL
  • GH-BP truncated form of the GHR
  • GH-BP encompasses the extracellular domain of the receptor, and could be the result of proteolytic cleavage of the native receptor or alternative RNA splicing. It has been
  • GH levels that is to say, the less GH-BP, the more serum GH present; Amit et al., 1992,
  • GH relatively more active in the GH receptor assay and therefore contribute negatively to
  • the present invention relates to methods and compositions for inhibiting
  • the present invention provides for a prolactin
  • prolactin variant in inhibiting the proliferation of a cell which expresses a prolactin
  • the invention is based in the observation that a prolactin variant is capable of
  • prolactin variant was able to induce apoptosis in cancer cells.
  • the prolactin variant is a mutated form of human prolactin in which the glycine amino
  • the glycine at position 129 of human prolactin is substituted with arginine.
  • the present invention provides for a
  • prolactin binds to its receptor.
  • variants and truncated prolactin receptors of the invention may be used in methods of
  • the present invention further provides methods for inducing apoptosis in
  • the invention is based on the observation that a
  • prolactin variant is capable of inducing cellular apoptosis in human breast cancer cells.
  • the present invention provides
  • tamoxifen include, but are not limited to, tamoxifen, raloxifene, or ICI 164384 (Imperial Chemical
  • the method is based on the observation that the administration of a prolactin
  • a prolactin variant may be used in conjunction with an anti-
  • anti-androgens include ,but are not limited to, fiutamide, anandron or
  • the present invention may be used in the treatment
  • FIGURE 1A Schematic representation of the cloning and construction of
  • FIGURE IB Plasmid map and general strategy of PCR-directed
  • pcDNA3 the parental vector, contains human immediate-early
  • CMV cytomegalovirus
  • bovine GH gene BGH pA
  • cDNA was cloned using RT-PCR from human pituitary mRNA and inserted into BstXl
  • Mutation was generated by designing PCR primers at Xba I sites.
  • FIGURE Data from competitive radioreceptor binding experiments for
  • HTB 123 HTB 123
  • T47D are human breast cancer cell lines.
  • the y axis represents the percent specific
  • FIGURE 3 Western blot analysis showing phosphorylation of STAT
  • lane 1 depicts a control culture
  • lane 2 depicts a culture
  • lane 3 depicts a culture receiving 5 nM of hPRLA
  • lane 4 depicts the competitive effects when the culture is exposed to 5 nM of hPRL and 5 nM of
  • hPRLA hPRLA
  • lane 5 depicts the competitive effects when the culture is exposed to 5 nM
  • FIGURE 4 Effects of growth hormone and prolactin on breast cancer cell
  • the x-axis represents the concentration of hGH or hPRL present in the
  • the y axis represents the total cell
  • FIGURE 5A-B Effects of various concentrations of hPRL or the
  • G129R prolactin variant hPRLA on the proliferation of T47D human breast cancer cells
  • FIGURE 6 Diagram of a mixed cell culture assay for evaluating the
  • FIGURE 7 Effects of recombinantly expressed hPRL (L-PRL) and the
  • FIGURE 8 Effects of recombinantly expressed hPRL (L-PRL) and the
  • FIGURE 9A-B Proliferation of either (A) T47D human breast cancer
  • FIGURE 10A-B Amino acid sequences of various human and non-reacted amino acids
  • FIGURE 11 Schematic illustration of the mechanism of GH or hPRL
  • Arg represents the substitution mutation in the third ⁇ -helix resulting in hindering a
  • FIGURE 12 Immunoblot analysis of hPRL-G129R gene expression by
  • Lanes A-D represent samples containing purified hPRL (from NIH) as
  • Lanes E-H represent culture media from stably transfected mouse L cells.
  • FIGURE 13 Antagonistic effects of hPRL-G129R on tyrosine
  • Lane assignments are A, negative control; B, cells stimulated with 100 ng/ml hPRL;
  • C cells treated with 100 ng/ml of hPRL-G129R
  • D cells treated with 10Q ng/ml of hPRl
  • FIGURE 14A-E Light microscopic examination of T47-D human breast
  • the x-axis represents the
  • FIGURE 16A-B Dose-response effects of 4-OH-Tamoxifen (17A) and
  • hPRl-G129R (17B).
  • the x-axis represents the concentration of 4-OH-Tamoxif ⁇ n (17A)
  • FIGURE 17 Dose-response inhibitory effects of hPRL-G129R on hPRL
  • the x-axis represents the concentration of hPRL-
  • FIGURE 18 Dose-response inhibitory effects of hPRL-G129R and its
  • the x-axis represents the hPRL-G129R concentration either in th ⁇ absence (open
  • FIGURE 19A-B Dose-response inhibitory effects of hPRl-G129R in two
  • the x-axis represents the co-
  • FIGURE 20A-F Dose response of T-47D human breast cancer cells to
  • FIGURE 21A-E Time course of T-47D human breast cancer cells
  • FIGURE 22A-H Response of multiple breast cancer cells to 4-OH-
  • FIGURE 23 A-F Response of multiple breast cancer cells to treatment
  • FIGURE 24 Induction of Caspase-3 by hPRL-G129R. The effect of
  • FIGURE 25 Response of two prostate cancer cells to treatment with
  • the present invention provides for prolactin (PRL) variants which
  • prolactin refers herein to human and nonhuman animal
  • prolactins include, but are not limited to, prolactins
  • prolactin (PRL) variant refers to a form of prolactin which has
  • sequence of the native form has been altered by the insertion, deletion, and/or substitution
  • PRL has a proliferative effect on a species of
  • a PRL variant according to the invention inhibits the proliferation of the species of
  • FIGURE 5 A illustrates a working example
  • hPRLA glycine at position 129 with an arginine residue
  • T47D cells relative to T47D cells lacking the added hPRL or hPRLA; it is believed that
  • T47D levels produce PRL (Ginsberg and Vonderharr, 1995, Cancer Res. 55:2591-2595).
  • a PRL variant may be identified as an
  • Z is less than Y and may be a negative
  • the PRL variant is a
  • the substituent amino acid may be neutral-polar
  • amino acids such as alanine, valine, leucine, isoleucine, phenylalanine, proline,
  • methionine neutral non-polar amino acids such as serine, threonine, tyrosine, cysteine,
  • tryptophan asparagine, glutamine, aspartic acid
  • acidic amino acids such as aspartic and
  • glutamic acid glutamic acid
  • basic amino acids such as arginine, histidine or lysine.
  • the glycine at position 129 of hPRL may be substituted
  • valine leucine, isoleucine, serine, threonine, proline, tyrosine, cysteine, methionine,
  • the present invention provides for a prolactin variant wherein
  • a prolactin variant is linked to
  • the prolactin is another protein as part of a fusion protein.
  • the prolactin is another protein as part of a fusion protein.
  • the prolactin is another protein as part of a fusion protein.
  • variant may be linked to interleukin 2.
  • interleukin 2 One nonlimiting example of such an embodiment
  • the PRL variants of the invention may be prepared by chemical synthesis
  • a cDNA of PRL may be prepared using
  • RNA or cDNA prepared from a cell which
  • PRL such as a pituitary cell
  • nucleic acid encoding the PRL variant may be incorporated into an expression vector
  • the expression vector may
  • a transcription termination site including a transcription termination site, a polyadenylation site, a ribosome binding site,
  • Suitable expression systems include mammalian cells, insect cells, etc.
  • Suitable expression vectors include herpes simplex viral based
  • vectors such as pHSVl (Geller et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:8950-8954); retroviral vectors such as MFG (Jaffee et al, 1993, Cancer Res. 53:2221-2226), and in
  • Moloney retroviral vectors such as LN, LNSX, LNCX, LXSN (Miller and
  • vaccinia viral vectors such as MVA (Sutter
  • AAV/neo Mura-Cacho et al., 1992, J. Immunother. 11 :231-237
  • lentivirus lentivirus
  • pCDNA3 and pCDNAl Insulin
  • pET 11a As pCDNA3 and pCDNAl (InVitrogen), pET 11a, pET3a, pETl Id, pET3d, pET22d, and
  • pET12a Novagen
  • plasmid AH5 which contains the SV40 origin and the adenovirus
  • a PRL variant produced in a recombinant expression system may then be
  • the present invention provides for cell-free truncated prolactin receptors
  • PRL-BP(s) PRL-BP(s)
  • a PRL-BP may be prepared by removing all or a part of the
  • transmembrane and/or intracellular domains of the PRLR either enzymatically or using
  • nucleic acid molecules encoding the native amino acids For recombinant preparation, nucleic acid molecules encoding the native amino acids
  • prolactin receptor may be prepared and then altered to encode a PRL-BP.
  • PRL-BP prolactin receptor
  • the PRLR may be cloned using techniques as set forth in
  • the methods may include in vitro recombinant DNA
  • fusion proteins that can facilitate, for example, solubility or
  • Such fusion proteins can be made by ligating the appropriate nucleic acid
  • proteins can comprise, for example, one or more of the extracellular domains or portions,
  • the ligand-binding portion preferably the ligand-binding portion.
  • expression vector such as pcDNA3.1/His Xpress (Invitrogen Corp., San Diego, CA) may
  • This vector contains a human immediate-early cytomegalovirus promoter and
  • bGH poly A addition signal.
  • it offers an in frame (His)6 peptide at the
  • hPRL-BP produced using such a vector in cell culture may be concentrated by
  • hPRL-BP following ultrafiltration may be determined by protein assay and confirmed by
  • a truncated PRL-BP may be made by protein synthesis
  • truncated PRL-BP may be prepared by purification of full length PRLR protein, from either naturally occurring or
  • proteolytic enzymes such as trypsin
  • the present invention provides a cell-based assay system that can be used
  • the cell-based assay system of the invention is designed to determine whether aberrant cell proliferation.
  • the assay system is based on the observation that the PRLR
  • G129R is capable of inducing apoptosis in cells expressing the PRLR.
  • a cell-based assay system is
  • Compounds that may affect PRLR activity include but are not
  • prolactin receptor activity comprises the following steps:
  • prolactin receptor has been identified.
  • cells that endogenously express PRLR can be used to screen
  • the cells are transformed cells, such as for example, breast cancer cells or prostate cancer cells.
  • cells that do not normally express PRLR can be used.
  • the cells expressing PRLR are
  • the cells are also added to the assay. After exposure, the cells can be assayed to
  • Assays designed to measure apoptosis include the
  • TUNEL terminal deoxynucleotidly transferase mediated dUTP nick end labeling
  • test compound The ability of a test compound to induce the level of apoptosis, above
  • test compound acts as an antagonist to inhibit signal transduction mediated by PRLR.
  • control indicates that the test compound induces signal transduction mediated by PRLR.
  • High throughput screening can be accomplished by plating the test cells
  • the wells will also contain complete medium, and in instances where an agonist
  • the cells are assayed for apoptosis using methods such
  • Potential antagonists are those compounds that induce
  • Potential agonists are those compounds that
  • Compounds may include, but are not limited to, peptides such as, for example
  • soluble peptides including but not limited to members of random peptide
  • variants of PRL may be screened for their ability to regulate the activity of the PRLR.
  • the present invention provides for methods and compositions whereby a
  • PRL variant which acts as a PRL antagonist
  • PRLR truncated form of the PRLR
  • PRL may be used to inhibit PRL-mediated cell proliferation.
  • method of the invention comprises the administration of a prolactin variant, or a truncated
  • truncated PRLR also referred to as a PRL-BP
  • PRL-BP truncated PRLR
  • PRLR and/or PRL so as to permit the inference of an effect which varies according to
  • PRL/PRLR availability For example, the activity of a hPRL variant or a truncated
  • hPRLR may be tested in all or a subset of the following five different human breast
  • T-47D 25,800/cell
  • MCF-7 8,300/cell
  • HTB19 (6,435/cell
  • HTB20 5,480/cell
  • HTB123 (1,094/cell
  • breast cancer cell lines is preferred over the use of the rat Nb2 T-cell lymphoma cell line,
  • the PRL variant or the truncated PRLR include (i) (for variant PRL) a competitive
  • receptor binding assay to examine if the antagonists are competing at the receptor level
  • proliferative or anti-pro liferative effects of PRL, variant PRL, or truncated PRLR is a
  • a PRL-BP of the invention include both benign and malignant proliferatiori of cells which
  • PRLR proliferative diseases
  • the breast including benign conditions such as breast adenomas and fibrocystic disease,
  • breast cancer including ductal, scirrhous, medullary,
  • prostate including benign prostatic hypertrophy and prostate cancer (local or metastatic).
  • Proliferative conditions involving cells which express a receptor homologous to the PRLR may also be treated, including conditions involving cells which express a growth
  • prolactin variants are capable of
  • the present invention provides methods for inducing apoptosis in cells expressing the
  • prolactin receptor as well as cells expressing a receptor homologous to the prolactin
  • expression of the PRLR receptor can be targeted to a specific cell population
  • nucleic acid molecules targeted for apoptosis, such as a cancer cell population.
  • expressing PRLR can be transferred into the targeted cell population using methods such
  • the receptor can be activated through contact with prolactin
  • the PRL variant or PRL-BP are in the treatment of proliferative conditions.
  • the PRL variant or PRL-BP are in the treatment of proliferative conditions.
  • condition to be treated is breast cancer
  • additional agents used in a combined regimen may include anti-estrogens such as
  • condition to be treated is prostate
  • additional agents used in a combination regimen may include an anti-androgen
  • a combined treatment regimen is based on the
  • compositions comprising a
  • PRL variant or PRL-BP in a suitable pharmaceutical carrier, for use in the foregoing
  • compositions may be administered by any suitable technique, including
  • compositions suitable for use in the present invention are provided.
  • an effective dose refers to that amount
  • PRL variant or PRL-BP required to inhibit proliferation of cells expressing the PRLR
  • the effective dose may be any one of the established in cell culture systems and/or in transgenic animals.
  • the effective dose may be any one of the established in cell culture systems and/or in transgenic animals.
  • cell proliferation assays are examples of cell proliferation assays.
  • assays may be performed to quantitate the
  • Inhibition of tumor cell growth can be assayed to detect PRL variant or PRL-BP
  • PRL variant or PRL-BP is that amount required to inhibit the proliferation of cancer cells
  • BP in conjunction with, one or more, additional agent.
  • agents include, for example. anti-estrogens, such as tamoxifen, or anti-androgens. Determination of effective amounts
  • the amount of the composition will, of course, also be dependent on the
  • Gly 129 of hPRL corresponds to Gly 120 of hGH and it is absolutely conserved among
  • RT/PCR was carried out using a kit from Perkin-Elmer
  • FIGURE 1 preparation of the pUCIG-Met expression vector, is summarized in FIGURE 1.
  • the parental plasmid which contains the hPRL cDNA and_a Ml 3 Fl
  • FIGURE 1 origin of replication was transformed into E. coli (CJ236).
  • annealing buffer 200 mM Tris-HCl, 20 mM MgCl 2 , 100 mM NaCl
  • the oligonucleotide (5'CGGCTCCTAGAGAGGATG-GAGCT3'), which encodes the G129R mutation
  • hPRLA hPRLA
  • A the "A” referring to its antagonist activity.
  • the hPRL and hPRLA-encoding nucleic acids were each inserted into a
  • hPRL and hPRLA mouse L cells [thymidine kinase-negative (TK) and adenine
  • APRT phosphoribosyl transferase-negative
  • hPRLA (-5-10 mg/l/24h/million cells) were prepared.
  • membranes were obtained from Amicon, Inc. (Northorough, MA). Two types of membranes were used, YMIO and YM100. A 200ml stirred cell with Amicon YM100
  • the permeate (>90% of recovery of hPRL) was applied onto a second
  • the concentration of hPRL or hPRLA was determined using
  • Radioreceptor binding assay Purified hPRL was labeled with Na 125 I by
  • Lactoperoxidase (10 ⁇ g dissolved in 10 ⁇ l of 0.4 mol liter acetate
  • the monolayer of cells was exposed to serum-free conditioned medium containing
  • STAT proteins represent a family of proteins, having molecular masses of
  • GHR or PRLR containing cells are treated with GH or PRL, respectively.
  • phosphorylation of STAT 5 is a receptor mediated event and is thought to be an important
  • hPRLA to inhibit induction of STAT 5 phosphorylation by wild type PRL.
  • HRP peroxidase
  • the human breast cancer cells were:
  • FIGURE 6 diagramatically represented in FIGURE 6.
  • breast cancer cells were co-reacted
  • T47D T47D were added to wells of a multi-well cell culture plate. In certain wells, which served as a control, no expressor cells were added. Then, increasing numbers of
  • control culture was subtracted. The resulting number could then be compared to the
  • FIGURE 2 demonstrate that two cell lines (T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D and HTB 123) among T-47D
  • leukemia cells leukemia cells, lymphoma cells and retinoblastoma cells.
  • FIGURE 3 shows that the induction of
  • FIGURE 4 The bell shaped dose response curves suggest that similar mechanisms (i.e.,
  • receptors are occupied by a single ligand via the high affinity site (the "self- antagonism"
  • FIGURE 5 A-B compares the effects of hPRL and hPRLA (the G129R
  • T47D cells (relative to untreated control cultures), hPRLA and tamoxifen had a
  • FIGURES 7 and 8 depict the results of mixed cell culture assays in which
  • hPRLA the G129R variant of human prolactin
  • breast cancer cells for 24 or 72 hours (FIGURE 7) or for one, two, three or five days
  • FIGURE 8 While hPRL resulted in an increase in T47D proliferation (relative to
  • hPRLA inhibited proliferation by up to 100 percent.
  • FIGURE 9A-B compares the inhibitory effects of hPRLA in mixed cell
  • hPRLA expressed by transfected L cells had an inhibitory effect on both cell lines, but the effect was greater on T47D cells, probably because there
  • hPRL-BP cDNA was cloned using reverse transcription (RT) followed by
  • the hPRL-BP antisense primer was designed at a
  • Ncol restriction enzyme cutting site which is located 66 bases from the putative
  • the sense primer was designed including
  • sequence hPRL-BP was determined by the dideoxy chain-termination method using
  • RT-PCR The RT-PCR technique was used to clone hPRL cDNA.
  • a RT-PCR kit was from Perkin-Elmer, Inc. (Norwalk, CT).
  • primer for the RT reaction was designed 2 bases from the stop codon (in bold) of hPRL
  • CMV immediate-early cytomegalovirus
  • This vector also contains
  • FIGURE 11 Oligonucleotide Directed Mutagenesis hPRL-G129R cDNA
  • mouse L fibroblast cell line were acquired from ATCC. Both human breast cancer cell
  • DMEM DMEM for MCF-7 and L cells
  • RPMI- 1640 medium RPMI- 1640 medium
  • Conditioned media containing hPRL and hPRL-G129R was prepared as
  • the concentration of hPRL or hPRL-G129R was determined by hPRL IRMA.
  • T47-D cells were plated in
  • Hybond-ECL membrane (Amersham, IL) at 100 volts constant voltage
  • HPRLG129R Conditioned Media. The assay conditions were modified
  • T47-D cells were trypsinized and passed into 96 well plates in RPMI- 1640 media
  • each cell line was pre-determined after titration assay.
  • T47-D cells 15,000 cells/well were plated. The cells were allowed to settle and adhere overnight (12-18 hours) and
  • the nucleotide sequence of hPRL was determined by the dideoxy chain-
  • HPRL-G129R cDNA was also generated by PCR and sequenced.
  • hPRL-G129R STAT proteins represent a family of proteins with a molecular mass of
  • FIGURE 13 suggesting that it is functioning as a hPRL antagonist.
  • hPRL-G129R completely inhibited STAT protein phosphorylation induce.d by nPRL.
  • hPRL-G129R were tested further for their ability to stimulate/inhibit breast cancer cell
  • FIGURES 15-18 96 well cell proliferation assay results are shown in FIGURES 15-18.
  • FIGURE 17 More importantly, when hPRL-G129R was applied together with 4-OH-
  • Tamoxifen resulted in a 15% inhibition, yet, in the presence of lOOng/ml of hPRL-G129R
  • MCF-7 cells was shifted to the right as compared to that of T47-D cells, i.e. it required
  • T47-D cells (Ormandy et al, Genes Dev. 15:167-178; Shih, 1981, In: Hormones and
  • the human breast cancer cell lines MDA-MB- 134, T-47D, BT-474 and
  • MCF-7 were obtained from ATCC. These breast cancer cell lines were chosen based on
  • the cell line MDA-MB- 134 has the highest PRLR
  • T-47D cells obtained from ATCC were grown in RPMI
  • MCF-7 cells were grown in DMEM medium (phenol red
  • the MDA-MB- 134 cells were grown in
  • the breast cancer cells were trypsinized (0.02% Trypsin - EDTA) and
  • the MDA-MB-134 VI cells were
  • TUNEL TUNEL labeling
  • the fluorescein-labeled dUTP is incorporated at the 3-OH ends by using the enzyme
  • the slide was examined under a FITC filter using an
  • Apoptosis (programmed cell death) is one of the central physiological
  • the PRLR antagonist G129R is able to induce
  • FIGURE 20A-F shows that G129R induced apoptosis in a dose dependent manner after 24 h treatment and that apoptosis occurs even at physiological concentrations (50 ng/ml,
  • FIGURE 20C In order to demonstrate the specificity of G129R to the PRLR, hPRL
  • MCF-7 or BT-474 cells at a concentration as high as 1 ⁇ M as assayed by the same
  • FIGURE 22A-H In contrast to 4-OH-Tamoxifen, 250 ng of G129R induced apoptosis
  • T-47D cells were treated with 250 ng/ml of
  • hPRL-G129R for 2h.
  • the assay was performed in the presence of DEVD-CHO (caspase- 3 inhibitor) to demonstrate that the Caspase-3 induction by hPRL-G129R is a specific
  • prolactin as a major growth factor and undergo apoptosis when deprived of it by
  • the continued mitogenic signal provided by hPRL may override existing

Abstract

L'invention concerne différentes variantes de prolactine humaine qui tiennent lieu d'antagonistes au niveau du récepteur de prolactine. L'invention concerne également l'utilisation desdites variantes pour le traitement du cancer et d'affections proliférantes chez l'homme, y compris les affections malignes et bénignes du sein et de la prostate.
PCT/US1999/010545 1998-05-12 1999-05-12 Utilisation d'agents antiprolactine pour le traitement d'affections proliferantes WO1999058097A2 (fr)

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AU43086/99A AU4308699A (en) 1998-05-12 1999-05-12 Use of anti-prolactin agents to treat proliferative conditions

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US8512898P 1998-05-12 1998-05-12
US60/085,128 1998-05-12

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007047803A2 (fr) * 2005-10-20 2007-04-26 Ghc Research Development Corporation Utilisation de la prolactine dans le traitement prophylactique du cancer
JP2008178410A (ja) * 2007-01-25 2008-08-07 F Hoffmann La Roche Ag バナジウム含有ホスファターゼ阻害剤の増強
EP1949894A3 (fr) * 2007-01-25 2011-12-28 Roche Diagnostics GmbH Amélioration des inhibiteurs de phosphatase contenant du vanadium

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007047803A2 (fr) * 2005-10-20 2007-04-26 Ghc Research Development Corporation Utilisation de la prolactine dans le traitement prophylactique du cancer
WO2007047803A3 (fr) * 2005-10-20 2007-09-07 Wen Yuan Chen Utilisation de la prolactine dans le traitement prophylactique du cancer
JP2008178410A (ja) * 2007-01-25 2008-08-07 F Hoffmann La Roche Ag バナジウム含有ホスファターゼ阻害剤の増強
US8008035B2 (en) 2007-01-25 2011-08-30 Roche Diagnostics Operations, Inc. Enhancement of vanadium-containing phosphatase inhibitors
EP1949894A3 (fr) * 2007-01-25 2011-12-28 Roche Diagnostics GmbH Amélioration des inhibiteurs de phosphatase contenant du vanadium
US8278086B2 (en) 2007-01-25 2012-10-02 Roche Diagnostics Operations, Inc. Enhancement of vanadium-containing phosphatase inhibitors
US8563263B2 (en) 2007-01-25 2013-10-22 Roche Diagnostics Operations Inc. Enhancement of vanadium-containing phosphatase inhibitors

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