Identification of an AP1-ZFP36 Regulatory Network Associated with Breast Cancer Prognosis.

It has been established that ZFP36 (also known as Tristetraprolin or TTP) promotes mRNA degradation of proteins involved in inflammation, proliferation and tumor invasiveness. In mammary epithelial cells ZFP36 expression is induced by STAT5 activation during lactogenesis, while in breast cancer ZFP36 expression is associated with lower grade and better prognosis. Here, we show that the AP-1 transcription factor components, i.e. JUN, JUNB, FOS, FOSB, in addition to DUSP1, EGR1, NR4A1, IER2 and BTG2, behave as a conserved co-regulated group of genes whose expression is associated to ZFP36 in cancer cells. In fact, a significant down-modulation of this gene network is observed in breast, liver, lung, kidney, and thyroid carcinomas compared to their normal counterparts. In breast cancer, the normal-like and Luminal A, show the highest expression of the ZFP36 gene network among the other intrinsic subtypes and patients with low expression of these genes display poor prognosis. It is also proposed that AP-1 regulates ZFP36 expression through responsive elements detected in the promoter region of this gene. Culture assays show that AP-1 activity induces ZFP36 expression in mammary cells in response to prolactin (PRL) treatment thorough ERK1/2 activation. These results suggest that JUN, JUNB, FOS and FOSB are not only co-expressed, but would also play a relevant role in regulating ZFP36 expression in mammary epithelial cells.

TNF-alpha enhances estrogen-induced cell proliferation of estrogen-dependent breast tumor cells through a complex containing nuclear factor-kappa B.

Breast tumors are usually classified according to their response to estrogens as hormone-dependent or -independent. In this work, we investigated the role of the proinflammatory cytokine TNF-alpha on the estrogen-receptor-positive T47D breast ductal tumor cells. We have found that TNF-alpha exerts a mitogenic effect, inducing cyclin D1 expression and activation of the transcription factor NF-kappaB. Importantly, activation of NF-kappaB was required for estrogen-induced proliferation and cyclin D1 expression. TNF-alpha enhanced the estrogen response by increasing the levels and availability of NF-kappaB. Chromatin immunoprecipitation analysis suggested that the action of estrogens is mediated by a protein complex that contains the activated estrogen receptor, the nuclear receptor coactivator RAC3 and a member of the NF-kappaB family. Finally, our results demonstrate that activation of this transcription factor could be one of the key signals for estrogen-mediated response.

Evidence for the transforming activity of a truncated Int6 gene, in vitro.

Int6/eIF3-p48 was first identified as a common integration site for MMTV in mouse mammary tumors. In all cases, the MMTV integration event resulted in an interruption of the normal Int6 transcript from one allele leaving the second allele intact and operative. We hypothesize that insertion of MMTV into Int6 results in a mutated allele that encodes a shortened Int6 mRNA and protein (Int6sh), which either modifies normal Int6 function or possesses a new independent function. To confirm the transforming potential of the mutation and its dominant function, we transfected two mammary epithelial cell lines, MCF10A (human), and HC11 (mouse), with Int6sh under the control of the elongation factor-1alpha (eEF1A) promoter. Expression of Int6sh in MCF10A and HC11 mammary epithelial cells leads to anchorage-independent growth in soft agar indicative of a transformed phenotype. Colonies selected from agar exhibited high levels of mutated Int6sh and wild type Int6 RNA transcripts by RT-PCR and Northern blot analysis. In addition, Int6sh transformed MCF10A and HC11 cells formed nodular growths, in vivo, in immune compromised hosts. NIH3T3 cells, mouse embryo fibroblasts, were also transformed to anchorage-independent growth in vitro by Int6sh expression. These observations provide direct evidence that the Int6 mutations observed in MMTV-induced tumors and hyperplasia contribute to the malignant transformation of the mammary epithelial cells.

Impairment of mammary lobular development induced by expression of TGFbeta1 under the control of WAP promoter does not suppress tumorigenesis in MMTV-infected transgenic mice.

It has previously been shown that transgenic female mice expressing TGFbeta1 under control of regulatory elements of the whey-acidic protein (WAP) gene were unable to lactate. This was due to the increased apoptosis of the cells committed to the lobular-lactogenic phenotype. Our goal was to determine whether the expression of WAP-TGFbeta1 transgene could inhibit MMTV (mouse mammary tumor virus) tumorigenic activity in the mammary gland. It is well known that the infection with this virus produces focal hyperplastic secretory nodules (HANs) and, some variants can also induce ductal pregnancy-dependent lesions (plaques). In either case, MMTV infection leads ultimately to the appearance of malignant mammary tumors. The results shown herein demonstrate that TGFbeta1 expression in the secretory mammary epithelium does not suppress mammary tumorigenesis in MMTV infected mice. Although MMTV infected WAP-TGFbeta1 transgenic females displayed a strong impairment of lobule-alveolar development, carcinogenesis induced by any of the four MMTV variants used herein proceeded unabated. WAP-TGFbeta1 tumors that showed a strong expression at the WAP promoter, appeared later and grew more slowly than their wild-type counterparts. Transgenic females also had a lower incidence of HANs and plaques. Our study suggests that the epithelial target cells for tumorigenic mutations are probably progenitor cells that are not susceptible to the apoptotic effect of TGFbeta1. Alternatively, their daughters cells that display the secretory phenotype and could be more involved in the formation of premalignant lesions continue to die due to the expression of the transgene.

Isotopic labelling studies on far-infrared spectra of nickel-histamine complexes.

Nickel-histamine (hm) complexes type [Ni(hm)Cl2] and [Ni(hm)3]X2 (Where X= Cl, Br, I, ClO4) were investigated in the far-infrared region. Metal isotope labelling and deuteration effects were employed for observed band assignments. Metal-ligand vibrations were discussed and correlated with the structures of the complexes.

Isotopic studies of the metal-ligand vibrations in histamine complexes with copper(II).

Two known copper complexes of the formulae Cu(hm)2(ClO4)2, Cu(hm)Cl2 and new, Cu(hm)Br2 (hm = histamine) have been investigated in the 600-50 cm(-1) far-infrared region. Assignments of vibrations related to metal-ligand bonds have been made based on metal isotope substitution, partial deuteration and halogen sensitivity. Copper-hm stretching vibrations have been localised at 420, 417 and 411 cm(-1) for modes involving amine nitrogen and at 282, 270, 259 cm(-1) for those of imidazole nitrogen. Vibrational coupling between some metal-ligand modes has been postulated.

An entire functional mammary gland may comprise the progeny from a single cell.

Any epithelial portion of a normal mouse mammary gland can reproduce an entire functional gland when transplanted into an epithelium-free mammary fat pad. Mouse mammary hyperplasias and tumors are clonal dominant populations and probably represent the progeny of a single transformed cell. Our study provides evidence that single multipotent stem cells positioned throughout the mature fully developed mammary gland have the capacity to produce sufficient differentiated progeny to recapitulate an entire functional gland. Our evidence also demonstrates that these stem cells are self-renewing and are found with undiminished capacities in the newly regenerated gland. We have taken advantage of an experimental model where mouse mammary tumor virus infects mammary epithelial cells and inserts a deoxyribonucleic acid copy(ies) of its genome during replication. The insertions occur randomly within the somatic genome. CzechII mice have no endogenous nucleic acid sequence homology with mouse mammary tumor virus; therefore all viral insertions may be detected by Southern analysis provided a sufficient number of cells contain a specific insertional event. Transplantation of random fragments of infected CzechII mammary gland produced clonal-dominant epithelial populations in epithelium-free mammary fat pads. Serial transplantation of pieces of the clonally derived outgrowths produced second generation glands possessing the same viral insertion sites providing evidence for self-renewal of the original stem cell. Limiting dilution studies with cell cultures derived from third generation clonal outgrowths demonstrated that three multipotent but distinct mammary epithelial progenitors were present in clonally derived mammary epithelial populations. Estimation of the potential number of multipotent epithelial cells that may be evolved from an individual mammary-specific stem cell by self-renewal is in the order of 10(12)-10(13). Therefore, one stem cell might easily account for the renewal of mammary epithelium over several transplant generations.

Expression of a truncated Int3 gene in developing secretory mammary epithelium specifically retards lobular differentiation resulting in tumorigenesis.

Insertional mutation of the Int3 gene, a member of the Notch gene family, is frequently associated with primary mouse mammary tumors induced by the mouse mammary tumor virus (MMTV). A major consequence of these mutations is the production of a shortened 2.4-kb tumor specific Int3 RNA transcript that encodes the entire intracellular domain of the Int3 protein. Previous studies have demonstrated that mammary gland development and function was severely impaired in transgenic mice expressing the truncated Int3 gene product from the MMTV viral promoter. Both mammary ductal growth and secretory lobule development were curtailed in these mice. These results were attributed to a gain of function modification of the Int3 gene, which led to a restriction of cell fate selection in the affected mammary epithelial cells. To confirm and extend these findings, truncated Int3 was expressed from the whey acidic protein (WAP) promoter, the activity of which, unlike that of the MMTV long terminal repeat, is restricted to the secretory mammary epithelial population. In transgenic mice carrying the WAP/Int3 construct, mammary ductal growth was unaffected in virgin females, but growth and differentiation of secretory lobules during gestation was profoundly inhibited. Coincidental with the block in lobular secretory differentiation, mammary dysplasia and tumorigenesis occurred in all breeding females by 25 weeks of age. In nonbreeding WAP/Int3 females, mammary tumor incidence also reached 100%, but only after 70 weeks. The WAP/Int3 mammary tumors were highly malignant, and most tumor-bearing females, irrespective of breeding history, developed metastatic lung lesions. These results suggest that WAP promotor-targeted Int3 function is associated with mammary secretory cell differentiation and maintenance in this transgenic model. Consistent with the conclusion that WAP-driven truncated Int3 expression influenced only lobular differentiation and not ductal growth and extension during mammary gland development, transplants of WAP/Int3 gland into nontransgenic mammary fat pads produced complete mammary ductal outgrowths in virgin FVB/N mice but failed to develop secretory lobules when the females were impregnated.

A novel non-mouse mammary tumor virus activation of the Int-3 gene in a spontaneous mouse mammary tumor.

In a mouse mammary tumor model system in which carcinogenic progression can be investigated, we have found a unique mutation of Int-3 associated with progression from premalignant lobular hyperplasia to tumor. Sequence analysis of the rearranged fragment revealed an insertion of an intracisternal type A particle (IAP) within the Int-3 gene. Int-3 is mutated frequently in mouse mammary tumor virus (MMTV)-induced mammary tumors by insertion of MMTV proviral DNA into this intragenic region. In these mutations, the insertion produces a chimeric Int-3 transcript encoding the cytoplasmic portion of the Int-3 protein driven by the MMTV long terminal repeat promoter. In this case, the IAP DNA was inserted in the opposite transcriptional orientation relative to Int-3; nevertheless, a similar chimeric RNA transcript driven by a cryptic promoter in the oppositely oriented 5' IAP long terminal repeat was generated. This is the first demonstration that an insertional mutation unrelated to MMTV activates an Int gene commonly associated with mammary tumorigenesis.

Transforming growth factor-alpha promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells.

Transforming growth factor (TGF)-alpha stimulates the growth and development of mammary epithelial cells and is implicated in the pathogenesis of human breast cancer. In this report we evaluate the consequences of overexpressing TGF-alpha in the mammary gland of transgenic mice and examine associated cellular mechanisms. When operating on a FVB/N genetic background (line MT100), TGF-alpha induced the stochastic development of mammary adenomas and adenocarcinomas f secretory epithelial origin in 64% of multiparous females. In contrast, tumors were exceedingly rare in virgin MT100 females, MT100 males, and multiparous FVB/N females. In MT100 females multiple foci of hyperplastic secretory lesions preceded the development of frank tumors; these initial lesions appeared during the involution period after the first lactation. Serial transplantation of these hyperplasias indicated an absence of proliferative immortality. Nevertheless, they gave rise to tumors at a low frequency and after a prolonged latency in virgin hosts; in multiparous hosts, tumors developed earlier and at a high incidence. The TGF-alpha transgene was highly expressed in hyperplasias and tumors but not in virgin and nonlesion-bearing tissue, suggesting that TGF-alpha overexpression provides a selective growth advantage. TGF-alpha also induced at lactation a 6.4-fold increase in DNA synthesis in MT100 epithelial cells, many of which were binucleated. MT100 mammary tissue experienced an obvious delay in involution, resulting in the postlactational survival of a significant population of unregressed secretory epithelial cells. In contrast, another line of transgenic mice on a CD-1 genetic background (MT42), in which TGF-alpha overexpression induced liver but not mammary tumors, failed to demonstrate postlactational epithelial cell survival. These data show that TGF-alpha promotes mammary tumorigenesis in multiparous MT100 mice by stimulating secretory epithelial cell proliferation during lactation and prolonging survival during involution. These points support the notion that TGF-alpha can act as a mitogen and also as a differentiation factor in mammary epithelium.

Detection and location of amphiregulin and Cripto-1 expression in the developing postnatal mouse mammary gland.

Amphiregulin (Ar) and Cripto-1 (Cr-1) are growth promoting peptides that share amino acid sequence homology with epidermal growth factor (EGF). The present study examined Ar and Cr-1 mRNA and protein expression during various stages of C57BL/6 mouse mammary morphogenesis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect transcripts for Ar and Cr-1 at all stages of mammary development. Immunocytochemical (ICC) localization demonstrated that in virgin 4-week to mature 12-week-old mouse fourth inguinal mammary gland, Ar and Cr-1 are expressed in the stromal cells, luminal epithelial cells, and myoepithelial cells of the branching ducts. Ar, and to lesser extent Cr-1, were also found in the epithelial cap cells and in the luminal epithelial cells of the advancing terminal end bud (TEB) from virgin 4-week and 6-week-old mice. Western blot analysis demonstrated that both Ar (28 and 26 kDa) and Cr-1 (90, 67, 56, and 21 kDa) proteins are expressed in virgin, 13.5 day midpregnant and in the 14 day lactating mammary gland. In addition, Ar and Cr-1 are associated with developing alveolar structures as determined by ICC. These results imply that together with EGF and transforming growth factor alpha (TGF alpha), Ar and Cr-1 may play salient roles as modifiers in the morphogenesis and differentiation of the mammary gland.

Ectopic TGF beta 1 expression in the secretory mammary epithelium induces early senescence of the epithelial stem cell population.

An important feature of the mammary gland is the regenerative capacity of its epithelium which is demonstrated upon successive cycles of lactation and involution. Pregnant mice expressing a whey-acidic protein (WAP) promoter-driven transforming growth factor-beta 1 (TGF beta 1) cDNA are unable either to generate a secretory mammary epithelium or to lactate. Here we investigate whether ectopic TGF beta 1 induces this phenotype by affecting the transgenic epithelium directly or in trans. Reciprocal transplantation of mammary tissue between normal and transgenic hosts resulted in the development of the respective phenotypes of the transplants within the same mammary fat pad. When isolated mammary epithelial cells from both were mixed before implantation so that transgenic and normal epithelium would develop together more proximately, both phenotypes were simultaneously observed in the resultant chimeric mammary outgrowths. Since no trans effect was detectable, we hypothesize that early expression of the transgene results in compromised lobular progenitor cells through an intracrine mechanism. Consistent with this posit, WAP promoter-driven protein expression was detected in individual cells of the subtending ducts of immature females at estrus. Transplantation of WAP-TGF beta 1 mammary gland into nonpregnant hosts revealed that transgenic implants, even those from young postpubertal virgin females, had a diminished ability to repopulate epithelium-free mammary fat pads. Accordingly, the ectopic expression of WAP-TGF beta 1 not only impairs lobular progenitors, but also promotes an early senescence of the regenerative capacity of the mammary ductal epithelium. This leads us to propose that mammary epithelial stem cells give rise to two functionally distinct progenitor cells in the mammary gland epithelium: one capable of producing daughters committed to ductal formation, the other capable only of producing daughters committed to lobular function.

Effect of sialoadenectomy on medroxyprogesterone-acetate-induced mammary carcinogenesis in BALB/c mice. Correlation between histology and epidermal-growth-factor receptor content.

To evaluate the possible involvement of the salivary glands in the modulation of medroxyprogesterone (MPA)-induced mammary tumorigenesis, 48 sialoadenectomized virgin BALB/c female mice and 47 controls were treated with 40mg MPA depot s.c. every 3 months for 1 year. Mammary tumors developed in 11 sialoadenectomized and in 34 control mice with similar latencies. In both groups, 75% of the tumors were ductal and progestin-dependent (PD) while the remainder were lobular and progestin-independent (PI). Epidermal growth factor (EGF) levels were measured in salivary glands (SG-EGF) and serum (S-EGF) in both groups. MPA induced a significant increase in SG-EGF and in S-EGF that became evident only after 1 month of MPA treatment. No increase in S-EGF was detected in MPA-treated sialoadenectomized mice, indicating that salivary glands are the major source of S-EGF. The presence of EGF receptors (EGF-R) was investigated in ductal PD and PI tumor lines and compared with 8 PI tumor lines of lobular origin. A significant difference in EGF-R content was found between lobular and ductal tumors. No increase in EGF-R was noted when ductal tumors became autonomous. EGF-R did not correlate with tumor growth rate and there was an inverse correlation between EGF-R and steroid receptors. When the effect of sialoadenectomy on tumor growth was tested in vivo in syngeneic transplants of 2 ductal PD, 1 ductal PI and 2 lobular PI mammary adenocarcinomas, it was not found to be significant when compared with the controls. It may be concluded that SG-EGF plays an important role in the induction of mammary adenocarcinomas by MPA, while it has no significant effect on the growth of established tumors.

Progesterone induction of mammary carcinomas in BALB/c female mice. Correlation between progestin dependence and morphology.

We have demonstrated that medroxyprogesterone acetate (MPA), when administered in high doses, induces mammary carcinomas in virgin female BALB/c mice. Since one of the possible explanations for this effect was its progestagenic effects, we decided to investigate whether progesterone (Pg) alone could also induce mammary adenocarcinomas in our model and if MPA at doses lower than those used to establish the model was also carcinogenic. A total of 136 mice were subdivided into 3 groups: Group 1, 44 mice were implanted s.c. with 40 mg Pg silastic pellets at the beginning of the experiment, and 6 months later with a 20 mg Pg pellet; Group 2, 45 mice were similarly treated with MPA pellets; Group 3, 47 mice were inoculated s.c. with 40 mg MPA every three months. At the end of 20 months, 9 animals had developed mammary tumors in Group 1, 18 in Group 2 and 34 in Group 3 (actuarial incidence = 28%, 58%, and 98%, respectively); tumor latency was similar in all groups: 46.2 +/- 13.1, 51.3 +/- 9.9, and 50.1 +/- 2.1 weeks, respectively. Seven (Group 1), 14 (Group 2), and 25 (Group 3) tumors were transplanted into syngeneic mice to determine progestin dependence. All tumors, except one from Group 1, were histologically characterized. In Group 1 (Pg 60 mg), 4 tumors (67%) were infiltrating lobular carcinomas and 2 were ductal carcinomas (33%). In Group 2 (MPA 60 mg), 2 tumors (14%) were lobular and 12 were ductal adenocarcinomas (86%) (Group 1 vs Group 2: p < 0.05), whereas in Group 3 (MPA 160 mg), 8 were lobular carcinomas (32%) and 17 were ductal carcinomas (68%). In syngeneic passages all lobular tumors behaved as progestin independent (PI) and ductal tumors as progestin dependent (PD). All ductal tumors, except one, expressed estrogen receptors (ER) and progesterone receptors (PR), whereas receptor expression was variable in lobular carcinomas. It can be concluded that Pg induces mostly lobular, PI mammary tumors in BALB/c female mice. The fact that most MPA-induced tumors are ductal and PD suggests that the two hormones use different carcinogenic pathways.

Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation.

Transforming growth factor-beta 1 (TGF-beta 1) possesses highly potent, diverse and often opposing cell-specific activities, and has been implicated in the regulation of a variety of physiologic and developmental processes. To determine the effects of in vivo overexpression of TGF-beta 1 on mammary gland function, transgenic mice were generated harboring a fusion gene consisting of the porcine TGF-beta 1 cDNA placed under the control of regulatory elements of the pregnancy-responsive mouse whey-acidic protein (WAP) gene. Females from two of four transgenic lines were unable to lactate due to inhibition of the formation of lobuloalveolar structures and suppression of production of endogenous milk protein. In contrast, ductal development of the mammary glands was not overtly impaired. There was a complete concordance in transgenic mice between manifestation of the lactation-deficient phenotype and expression of RNA from the WAP/TGF-beta 1 transgene, which was present at low levels in the virgin gland, but was greatly induced at mid-pregnancy. TGF-beta 1 was localized to numerous alveoli and to the periductal extracellular matrix in the mammary gland of transgenic females late in pregnancy by immunohistochemical analysis. Glands reconstituted from cultured transgenic mammary epithelial cells duplicated the inhibition of lobuloalveolar development observed in situ in the mammary glands of pregnant transgenic mice. Results from this transgenic model strongly support the hypothesis that TGF-beta 1 plays an important in vivo role in regulating the development and function of the mammary gland.

Estrogen inhibition of MPA-induced mouse mammary tumor transplants.

Estrogen compounds were used to treat mice bearing syngeneic transplants of medroxyprogesterone acetate(MPA)-induced BALB/c mammary adenocarcinomas. Both MPA-dependent and MPA-independent tumor lines were used. These lines expressed estrogen (ER) and progesterone receptors (PR). We demonstrate that different doses of estradiol benzoate (EB) and 17-beta-estradiol (E2) inhibit tumor growth and induce tumor regression in both MPA-independent and -dependent tumors, even in the presence of MPA or progesterone (P). EB was unable to induce regression of (ER-) hormone-independent tumor lines. A few MPA-dependent tumors became resistant to the estrogenic treatment; in subsequent passages some of these tumors retained their MPA-responsiveness, although estrogen sensitivity was not recovered.

Hormone dependence of a mouse mammary tumor line induced in vivo by medroxyprogesterone acetate.

The administration of MPA to virgin female BALB/c mice led to the development of mammary adenocarcinomas, which in further in vivo transplants gave rise to both MPA-dependent and MPA-independent lines. In this paper we chose one of the MPA-dependent lines with high contents of estrogen (ER) and progesterone (PR) receptors, and were able to demonstrate that a) the growth of these tumors could be manipulated by the administration or the withdrawal of the hormonal supply; b) PR were down-regulated in MPA-treated mice; c) progesterone had the same stimulatory effect as MPA on tumor growth; d) tumors did not grow in estrogen-treated mice; e) tumor growth was much lower in males than in females; f) the presence of the ovaries had a positive influence on tumor growth, even in the presence of MPA; g) the withdrawal of progestin pellets in ovariectomized mice usually led to complete remissions followed by regrowth of the tumors after several weeks; and h) the regrowing tumors maintained their steroid receptor pattern and (in 3 out of 4 cases) their hormone-dependent behavior in further passages.

Transforming growth factor-beta activities in 'in vivo' lines of hormone-dependent and independent mammary adenocarcinomas induced by medroxyprogesterone acetate in BALB/c mice.

We have determined the presence of transforming growth factor-beta (TGF-beta)-like polypeptides in mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA) in BALB/c mice. In hormone-dependent tumors (HD) from nontreated and MPA-treated mice a high molecular weight (43 kDa) transforming activity was purified by Bio-Gel P-60 chromatography. This TGF was able to confer the neoplastic phenotype on NRK-49F cells without the addition of epidermal growth factor (EGF), though its activity was potentiated by EGF. It did not compete for binding to the EGF receptor, had no mitogenic activity on monolayer cultures of NRK fibroblasts, and was a potent inhibitor of DNA synthesis induced in these cells by EGF and insulin. In HD and hormone-independent tumors (HI) another TGF with a Mr of 13 kDa was isolated. This transforming activity showed the same biological properties as 43 kDa TGF, with the exception that in the absence of EGF it did not stimulate soft agar growth of NRK-49F cells. The synthesis of both factors in 'in vivo' HD tumors seems to be under MPA control, since it is much lower in HD tumors from MPA-treated mice. Further purification of the 13 and 43 kDa TGFs by hydrophobic interaction HPLC demonstrated that each one eluted in a different position, and that their elution profile differed from the TGF-beta from human platelets. The biological activity of the 13 and 43 kDa TGFs was not neutralized by a specific anti-TGF-beta antibody.

Progesterone receptors in estrogen-induced fibromatosis of guinea pigs.

The presence of estrogen and progesterone receptors was investigated in estrogen-induced guinea pig fibromatosis. These lesions were composed of fibroblast-like cells embedded in abundant collagen which stained positively for desmin and vimentin suggesting muscler and fibroblast differentiation. Cytosolic progesterone receptors ranging from 13 to 2045 fmol/mg protein were present in 10/13 samples while nuclear progesterone receptors were detected in 7/10 samples with values ranging from 8 to 4639 fmol/mg DNA. The Kd of the cytosolic progesterone receptor measured by a Scatchard analysis yielded a value of 3.35 X 10(-9) M with a sedimentation coefficient of 9S as determined by a sucrose gradient. Estrogen nuclear binders were found in 9/10 samples ranging from 6 to 282 fmoles/mg DNA and cytosolic estrogen binders in 10/13 with values of 9 to 349 fmoles/mg protein. The Kd of the cytosolic estrogen binder was 22 X 10(-9) M. This binder was not detected by a commercially available monoclonal antibody. Estrogen cytosolic binders and progesterone cytosolic receptors were also detected in some samples of non tumoral fibrotic tissue. It is concluded that this experimental model is appropriate for the investigation of the effects of estrogen and progesterone on proliferative fibroblastic lesions.

Mammary adenocarcinomas induced by medroxyprogesterone acetate: hormone dependence and EGF receptors of BALB/c in vivo sublines.

Mammary adenocarcinomas were induced by medroxy-progesterone acetate (MPA) in female BALB/c mice. From 5 primary tumors, 9 different sublines were established by s.c. transplantation into syngeneic female mice; these developed after a long latent period (4-12 months). Each subline was transplanted both into 4 mice treated with 40mg of MPA depot (s.c. contralaterally to the tumor inoculum) and into 4 non-treated mice. Of the 9 sublines, 6 proved to be hormone-dependent (MPA-D) and 3 hormone-independent or autonomous (MPA-I). However, even the autonomous lines, when treated with MPA, showed a slight increase in growth. All MPA-D lines had a high content of ER (20-254 fmoles/mg of protein), PR (63-710), PRL-R (44-74) and low or non-detectable EGF-R. Of the 3 MPA-I sublines that were studied, 2 showed a high content of ER (16-125), PR (27-708), PRL-R (19-70) and EGF-R (29-65) while the other one had a low content of ER (0-36), PR (0-13), no EGF-R and moderate PRL-R (15-52). Spontaneous mammary tumors of BALB/c and C3H origin, which also showed an MPA-I pattern of tumor growth, had high levels of EGF-R. We postulate that MPA has a direct effect on mammary tumor cells in MPA-D lines and that the expression of EGF-R is correlated with an autonomous pattern of growth.