Prolactin serum levels and breast cancer: relationships with risk factors and tumour characteristics among pre- and postmenopausal women in a population-based case-control study from Poland.

BACKGROUND: Previous prospective studies have found an association between prolactin (PRL) levels and increased risk of breast cancer. Using data from a population-based breast cancer case-control study conducted in two cities in Poland (2000-2003), we examined the association of PRL levels with breast cancer risk factors among controls and with tumour characteristics among the cases. METHODS: We analysed PRL serum levels among 773 controls without breast cancer matched on age and residence to 776 invasive breast cancer cases with available pretreatment serum. Tumours were centrally reviewed and prepared as tissue microarrays for immunohistochemical analysis. Breast cancer risk factors, assessed by interview, were related to serum PRL levels among controls using analysis of variance. Mean serum PRL levels by tumour characteristics are reported. These associations also were evaluated using polytomous logistic regression. RESULTS: Prolactin levels were associated with nulliparity in premenopausal (P=0.05) but not in postmenopausal women. Associations in postmenopausal women included an inverse association with increasing body mass index (P=0.0008) and direct association with use of recent/current hormone therapy (P=0.0006). In case-only analyses, higher PRL levels were more strongly associated with lobular compared with ductal carcinoma among postmenopausal women (P=0.02). Levels were not different by tumour size, grade, node involvement or oestrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2 status. CONCLUSIONS: Our analysis demonstrates that PRL levels are higher among premenopausal nulliparous as compared with parous women. Among postmenopausal women, levels were higher among hormone users and lower among obese women. These results may have value in understanding the mechanisms underlying several breast cancer risk factor associations.

Msx2 induces epithelial-mesenchymal transition in mouse mammary epithelial cells through upregulation of Cripto-1.

Epithelial-mesenchymal transition (EMT) is a process occurring during both embryogenesis and early stages of invasive cancer. Epithelial cells that undergo EMT become more migratory and invasive with a mesenchymal morphology. Herein we assess EMT induction in a mouse mammary epithelial cell line driven by Msx2, a homeobox-containing transcription factor important during mammary gland development. NMuMG cells, a normal mouse mammary epithelial cell line, stably transfected with a Msx2 cDNA showed downregulation of an epithelial marker E-cadherin and upregulation of the mesenchymal markers vimentin and N-cadherin. Furthermore, overexpression of Cripto-1, a member of the epidermal growth factor-CFC protein family already known to be involved in EMT, was detected in Msx2-transfected cells. The expression of Cripto-1 was accompanied by activation of the tyrosine kinase c-Src pathway and an increase in the invasive ability of the cells. Functional assays also demonstrated inhibition of the invasive behavior of the Msx2-transfected cells by a c-Src specific inhibitor. Moreover, immunohistochemistry of human infiltrating breast carcinomas showed positive staining for Msx2 only in the infiltrating tumor cells while the non-infiltrating tumor cells were negative. These results suggest that Msx2 may play a significant role in promoting EMT in epithelial cells that acquire properties involved in tumor invasion. J. Cell. Physiol. 219: 659-666, 2009. Published 2009 Wiley-Liss, Inc.

Lactose synthetase activity in mouse mammary glands is controlled by thyroid hormones.

Epithelial cells in explants from the mammary glands of euthyroid mature virgin mice are proliferatively dormant. They must undergo DNA synthesis and traverse the cell cycle in vitro before they are able to differentiate fully in response to insulin, hydrocortisone, and prolactin, and synthesize enzymatically active alpha-lactalbumin (measured as lactose synthetase activity). In contrast, glands from hyperthyroid mature virgin mice do not require DNA synthesis in vitro to differentiate. Explants from the euthyroid virgin tissue overcome their dependence on DNA synthesis when 10(-9) M 3,5,3'-triiodo-L-thyronine is added directly to the cultures in addition to the other three hormones. Explants from involuted mammary glands from euthyroid primiparous mice do not require DNA synthesis in vitro to make the milk protein even though they, like explants from mature euthyroid virgin tissue, are proliferatively dormant and do not contain detectable lactose synthetase activity in vivo. Glands from primiparous animals made mildly hypothyroid by ingestion of 0.1% thiouracil in drinking water during 7 wk of involution remain morphologically indistinguishable from glands of their euthyroid counterparts. However, explants from the glands of these hypothyroid animals revert to a state of dependence on DNA synthesis to differentiate functionally. These observations suggest that the dependence on DNA synthesis and cell cycle traversal for hormonal induction of lactose synthetase activity in the mouse mammary gland is controlled by thyroid hormones.

A role of estrogen/ERalpha signaling in BRCA1-associated tissue-specific tumor formation.

Estrogen and its receptor alpha (ERalpha) have been implicated in the tissue-specific tumorigenesis associated with BRCA1 mutations. However, the majority of breast cancers developed in human BRCA1 mutation carriers are ERalpha-negative, challenging the link between BRCA1 and estrogen/ERalpha in breast cancer formation. Using a mouse model lacking the full-length form of BRCA1, here we show that ERalpha is highly expressed in the premalignant mammary gland and initiation stages of tumorigenesis, although its expression is gradually diminished during mammary tumor progression. We demonstrate that the absence of full-length BRCA1 increases sensitivity of cells to estrogen-induced extracellular signal-regulated kinase 1/2 phosphorylation and cyclin D1 expression. The absence of BRCA1 turns the proliferation of ERalpha-positive cells from a paracrine fashion to an autocrine or endocrine fashion. Consequently, BRCA1-mutant cells are sensitized to estrogen-induced cell proliferation in vitro and mammary tumorigenesis in vivo. These findings illustrate a molecular mechanism for estrogen/ERalpha signals in BRCA1-associated tissue-specific tumor formation, and identify several key elements in the estrogen/ERalpha-signaling cascade that can serve as potential therapeutic targets for BRCA1-associated tumorigenesis.

Interlobular and intralobular mammary stroma: genotype may not reflect phenotype.

BACKGROUND: The normal growth and function of mammary epithelial cells depend on interactions with the supportive stroma. Alterations in this communication can lead to the progression or expansion of malignant growth. The human mammary gland contains two distinctive types of fibroblasts within the stroma. The epithelial cells are surrounded by loosely connected intralobular fibroblasts, which are subsequently surrounded by the more compacted interlobular fibroblasts. The different proximity of these fibroblasts to the epithelial cells suggests distinctive functions for these two subtypes. In this report, we compared the gene expression profiles between the two stromal subtypes. METHODS: Fresh normal breast tissue was collected from reduction mammoplasty patients and immediately placed into embedding medium and frozen on dry ice. Tissue sections were subjected to laser capture microscopy to isolate the interlobular from the intralobular fibroblasts. RNA was prepared and subjected to microarray analysis using the Affymetrix Human Genome U133 GeneChip. Data was analyzed using the Affy and Limma packages available from Bioconductor. Findings from the microarray analysis were validated by RT-PCR and immunohistochemistry. RESULTS: No statistically significant difference was detected between the gene expression profiles of the interlobular and intralobular fibroblasts by microarray analysis and RT-PCR. However, for some of the genes tested, the protein expression patterns between the two subtypes of fibroblasts were significantly different. CONCLUSION: This study is the first to report the gene expression profiles of the two distinct fibroblast populations within the human mammary gland. While there was no significant difference in the gene expression profiles between the groups, there was an obvious difference in the expression pattern of several proteins tested. This report also highlights the importance of studying gene regulation at both the transcriptional and post-translational level.

Requirement of essential fatty acids in the diet for development of the mouse mammary gland.

The ductal system of mammary glands in C3H mice that were started on diets deficient in essential fatty acid(s) (EFA) 1--2 weeks after weaning developed at a normal rate. The alveolar structures, however, began to disappear in the mice after 17 weeks on the EFA-deficient regimen. Injection sc of linoleic acid prevented the atrophic process. Alveoli were also absent in the glands of multiparous mice that were maintained for 32 weeks on the EFA-deficient diet. When the EFA-deficient regimen was started in females during midpregnancy and maintained continuously thereafter, both ductal and alveolar structures failed to develop in the mammary glands of the offspring. Linoleic acid appears to be required for development of ductal and alveolar structures during growth of the mammary gland and also for maintenance of the alveolar structures in the adult mammary gland.

Role of serum in the prolactin responsiveness of MCF-7 human breast cancer cells in long-term tissue culture.

MCF-7 human breast cancer cells, grown in long-term tissue culture, were found to be highly responsive to prolactin in terms of growth even in the presence of serum. Human prolactin, placental lactogen, and growth hormone (50-250 ng/ml) stimulated MCF-7 cells to grow when added to culture medium of cells in the presence of charcoal-stripped serum. Within 3 days of the hormone addition, a 4.4-fold increase in cell number was achieved with human prolactin at 100 ng/ml in the presence of 10% charcoal-stripped serum. Under these same conditions, estradiol-17 beta at 10(-8) M achieved only a 2-fold increase. After 6 days of culture, both estradiol-17 beta and prolactin gave a total 5-fold increase in cell number. No prolactin effect was achieved in the presence of 10% fetal bovine serum. Stripping fetal bovine serum with dextran-coated charcoal removes as much as 85% of the endogenous lactogens. Removal of these hormones is essential for demonstration of subsequent prolactin-induced growth response in MCF-7 cells, since bovine prolactin binds effectively to lactogen receptors on the surface of the cells but does not transmit a growth signal. When added simultaneously with human prolactin, bovine prolactin blocks the growth response to the former hormone. These results clearly demonstrate that, under the proper conditions of culture, the human breast cancer cell line MCF-7 is highly responsive to growth stimulation by homologous lactogenic hormones. This then affords us an excellent model for further studies on the possible role of prolactin in growth and maintenance of human breast cancer.

Prolactin synthesis and secretion by human breast cancer cells.

A possible autocrine function of prolactin (Prl) in human breast cancer was explored by the addition of a panel of anti-human Prl mAbs to T47Dco and MCF7 human breast adenocarcinoma cells. mAb 631 and mAb 390 inhibited cell growth by 86 and 68%, respectively, in the estrogen receptor-negative T47Dco cells and by 20 and 71%, respectively, in the estrogen receptor-positive MCF7 cells. Conditioned medium prepared from T47Dco cells was assessed for the presence of Prl-like molecules by its ability to stimulate growth of Prl-responsive Nb2 rat lymphoma cells. Growth of Nb2 cells under the influence of human Prl or conditioned medium was abolished when either solution was pretreated with mAb 390, followed by Immunobead precipitation (Bio-Rad, Melville, NY). T47Dco cells secrete 0.7 microgram lactogen/ml over a 24-48-h period. With the use of reverse transcription-PCR, an expected 612-bp band was detected by ethidium bromide staining, and its similarity to pituitary Prl was confirmed by Southern blot analysis with the use of human Prl cDNA as a probe. A single M(r) 22,000 band, the dominant size of monomeric pituitary Prl, was found in immunoprecipitates of both cell extracts and conditioned medium from T47Dco cells labeled metabolically with [35S]cysteine. These data suggest that human breast cancer cells synthesize and secrete biologically active Prl.

Effect of thyroid status on development of spontaneous mammary tumors in primiparous C3H mice.

Development of mammary tumors in primiparous C3H/HeN mice (mouse mammary tumor virus positive) in various thyroid states was followed for one year after removal of pups. Animals were either euthyroid or made hyperthyroid (by ingestion of thyroxine) or hypothyroid (by ingestion of 2-thiouracil) during involution. These manipulations resulted in significant changes in serum 3,5,3'-triiodo-L-thyronine and thyroxine levels without significant alterations in serum prolactin levels. At the end of one year postlactation, 90 to 86% of the euthyroid and hyperthyroid animals had developed mammary tumors, while the hypothyroid groups had only 70 to 72% tumor incidence. In two separate experiments, 50% tumor incidence was reached after 237 and 252 days in the hyperthyroid animals and after 242 and 252 days in the euthyroid groups. However, 50% tumor incidence in the hypothyroid groups was not reached until 290 and 287 days. The involuted mammary glands of all three groups were morphologically indistinguishable 10 weeks after removal of the pups. However, after 30 weeks, differences were seen. While glands from hyperthyroid and euthyroid animals retained a small degree of ductal branching with primitive alveoli, the glands from hypothyroid animals showed less ductal branching and were devoid of alveoli. Thus, the decrease in mammary tumor incidence in hypothyroid primiparous mice may be due to a greater degree of regression of the mammary epithelium in these animals.

Antiestrogen inhibition of prolactin-induced growth of the Nb2 rat lymphoma cell line.

The rat lymphoma cell line Nb2 is highly prolactin responsive in terms of growth. Estrogens are without effect in these cells that lack the estrogen receptor. The growth stimulation by lactogenic hormones is effectively inhibited by antiestrogens, such as tamoxifen and nafoxidine, at concentrations as low as 10(-10) M. The growth inhibition is partially reversed by replacement of the antiestrogens by prolactin. Nb2 cells contain estrogen-noncompetitive specific antiestrogen binding sites on their membranes that bind tamoxifen with an average Kd of 3.1 X 10(-10) M. Addition of antiestrogens to the binding reaction inhibits lactogenic hormone binding to membrane-bound receptors. The order of affinities of various antiestrogens (tamoxifen, nafoxidine, 2-(4-tert-butyl-phenoxy)ethyl diethylamine hydrochloride, and LY117018) for the antiestrogen binding sites parallels the order of their potencies as growth and lactogen binding inhibitors. These data suggest that antiestrogens, possibly acting through the antiestrogen binding sites, may function as antilactogens.

Binding of opioids to human MCF-7 breast cancer cells and their effects on growth.

The well characterized human breast cancer cell line, MCF-7, has been shown to possess membrane receptors for various opioid ligands, and these compounds have been shown to modulate the growth of the cells in culture. Using specific radioligands for the receptor types, we were able to demonstrate that the MCF-7 cells possess multiple opioid receptor types. Relatively high-affinity-binding sites are present for the mu- and kappa-specific ligands, while lower affinity sites are present for the delta-agonist. Opioid ligands specific for the different receptor types significantly inhibited the growth of the MCF-7 cells in a dose-dependent manner when grown in the presence of 10% fetal bovine serum. This inhibitory effect was reversed by the simultaneous administration of the opioid receptor antagonist, naloxone. However, the opioid effect appears to be restricted to the hormonally responsive fraction of the MCF-7 cell growth. Cells grown in the presence of charcoal-stripped fetal bovine serum are refractory to the effects of the opioids unless the media is supplemented with estradiol. The data presented here suggest an important regulatory role for opioids in the growth and development of human breast cancers.

Transplacental effects of diethylstilbestrol on mammary development and tumorigenesis in female ACI rats.

Female ACI rats were exposed to diethylstilbestrol (DES) transplacentally and followed to 10 months of age to assess the effect of the drug on mammary development and tumorigenesis. Pregnant rats were given injections of vehicle (sesame oil) or DES (total dose, 0.8 micrograms = low DES or 8.0 micrograms = high DES) on days 15 and 18 of gestation. Pellets containing 2.5 mg DES + 17.5 mg cholesterol (DES pellet) or 20 mg cholesterol (chol pellet) were implanted s.c. into 12-week-old female offspring, creating 6 experimental groups: vehicle exposure + chol pellet (1) or + DES pellet (2); low DES exposure + chol pellet (3) or + DES pellet (4); high DES exposure + chol pellet (5) or + DES pellet (6). At sacrifice, representative mammary tissue and all palpable mammary tumors were removed for histopathological analysis. Each of the 6 experimental groups contained a minimum of 32 rats from at least 14 litters. In computation of data, the unit of analysis was the litter. Groups which had received any DES (prenatally or postnatally) were found to have elongated nipples and enlarged pituitaries. The mammary gland whole mounts from all rats in groups 4 and 6 displayed extensive lobuloalveolar proliferation comparable to that seen in DES pellet controls (group 2). Mammary glands of approximately 75% of rats in groups 3 and 5 were categorized as showing the lowest grade of differentiation while this undifferentiated condition was seen in only 36% of group I controls. No palpable mammary tumors were found in rats exposed to vehicle in utero (group 1). But in group 5, a total of 6 tumors in 5 animals derived from 4 different litters were obtained, a difference shown to be statistically significant. Group 3 had 1 rat with 8 tumors. Among rats bearing the DES pellet, tumor latency was shortened significantly in both groups exposed to DES in utero. By 22 weeks after pellet implantation, 100% of the DES-exposed litters (groups 4 and 6) contained at least 1 tumor-bearing rat compared to about 50% of the tumor-bearing litters in group 2. Tumor multiplicity at sacrifice was increased significantly in the group exposed prenatally to the higher dose of DES. Histologically, the overwhelming majority of palpable mammary tumors from all tumor-bearing treatment groups were classified as adenocarcinomas. Prenatal exposure to DES did not alter the ratio of malignant to benign lesions observed, nor did it affect the degree of differentiation noted in the adenocarcinomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of pregnancy-specific genes in preneoplastic mouse mammary tissues from virgin mice.

Experimentally induced breast cancer is often preceded by the appearance of preneoplastic lesions which possess the attributes of hyperplastic normal tissue. These lesions can be isolated and carried as stably transplantable outgrowth lines which continue to morphologically resemble differentiating mammary tissue (Medina, D. Methods Cancer Res., 7: 3-53, 1973). We established seven serially transplantable hyperplastic alveolar nodule (HAN) outgrowth lines from virgin mouse mammary tissues following induction by mouse mammary tumor virus, dimethylbenz(alpha)-anthracene, and/or pituitary isografts. The expression of mammary differentiation-specific casein genes was measured in these hyperplastic outgrowths by immunocytochemistry, specific radioimmune precipitation, and blot hybridization of total RNA. All seven HAN outgrowth lines were immunologically positive for casein both in situ and upon explant culture. Unlike explants from normal virgin mouse mammary gland, exposure to insulin, hydrocortisone, and prolactin induced an increase in casein synthesis in HAN explant cultures which was independent of DNA synthesis. [35S]Methionine-labeled polypeptides synthesized in explant cultures of HAN outgrowths freshly isolated from virgin hosts were analyzed by radioimmune precipitation and gel electrophoresis. This analysis demonstrated that all major species of casein, alpha (Mr 46,000), beta (Mr 27,000), and gamma (Mr 25,000), were constitutively (i.e., in the absence of lactogenic stimuli) expressed in these preneoplastic alveolar mammary outgrowths. In support of this observation, RNA homologous to beta- and alpha-casein cDNA probes was often detectable in total RNA preparations from freshly isolated fragments of HAN outgrowths. A second mammary differentiation specific gene product, alpha-lactalbumin, was also detected in HAN outgrowths both in situ and following explant culture. Enzymatically active alpha-lactalbumin was present in extracts of freshly isolated HAN outgrowth tissues and was detectable in these same tissues by immunoperoxidase. In general, alpha-lactalbumin synthesis was increased during explant culture in the presence of lactogenic hormones; however, in contrast to casein synthesis, insulin-hydrocortisone-prolactin-induced increase in alpha-lactalbumin production in vitro was occasionally dependent upon DNA synthesis as it is in explants from normal virgin mouse mammary tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Difference between mammary epithelial cells from mature virgin and primiparous mice.

Mammary epithelial cells from mature virgin mice are similar to those from primiparous mice in several respects. However, there is one known difference. The cells from the mature virgin must traverse the cell cycle in order to become competent to make casein and enzymatically active alpha-lactalbumin in vitro; those from the primiparous animal can make these proteins without first traversing the cycle. In this regard, cells from human placental lactogen- and prolactin-treated mature virgins are, after involution, similar to those from primiparous mice. The developemental block in the cells from the mature virgin, imposed by preventing cell cycle traversal, has been partially delineated. It does not appear to reside at the levels of ultrastructural maturation or the formation of casein messenger RNA. Rather, the lesion is postranscriptional and may be at the level of translation, or posttranslational modification, or both.

Sensitivity of N-nitrosomethylurea-induced rat mammary tumors to cis-hydroxyproline, an inhibitor of collagen production.

The growth of primary N-nitrosomethylurea-induced rat mammary tumors was depressed by cis-hydroxyproline (CHP). This growth arrest appeared to be related to the ability of CHP to inhibit the deposition of basement membrane collagen as based on the following observations: (a) in vitro and in vivo, tumor cells synthesized type IV collagen, the collagen uniquely localized in basement membranes; (b) in vitro, the inhibition of tumor cell growth was preceded by a specific decrease in collagen accumulation with no effect on non-collagen protein synthesis; (c) a transplantable N-nitrosomethylurea-induced rat mammary tumor accumulated no type IV collagen as determined by polyacrylamide gel electrophoresis and indirect immunofluorescence. The growth of this tumor was not influenced by CHP; (d) an established human mammary tumor cell line, MCF-7, did not accumulate type IV collagen and was not inhibited by CHP. At the doses which effectively blocked the growth of primary N-nitrosomethylurea-induced mammary tumors, CHP and no toxic effects, and serum prolactin levels were not altered. The inhibitory effect was thus apparently due to the direct action of CHP upon the accumulation of collagen in cells which required type IV collagen production for continued growth.

Involvement of SHC, GRB2, SOS and RAS in prolactin signal transduction in mammary epithelial cells.

The peptide hormone prolactin (Prl) regulates proliferation of normal and malignant mammary cells. In the present study we demonstrate that two Prl responsive cell lines, NOG-8 and T47D, activate the JAK2-SHC-MAPK pathway in a rapid and transient manner. Within 1 min of Prl treatment there was an increase in association of JAK2 with SHC, followed by rapid phosphorylation of both the 52 kDa and 46 kDa SHC proteins. Grb2 and Sos associated with the SHC proteins within 1-3 min of Prl treatment in these mammary cells. Within 5 min of hormone treatment we observe an increase in ras-GTP suggesting activation of ras. We also showed a rapid and transient tyrosine phosphorylation of STAT5 in proliferating T47D cells which reached its peak after 30 min of Prl treatment. These results indicate that Prl receptors, after binding the ligand, activate several pathways for signal transduction leading to mitogenesis.

Purification and characterization of mouse alpha-lactalbumin from lactating mammary glands.

The whey protein, alpha-lactalbumin, was purified from lactating mammary glands of mice at high yields. It exists as two major charge forms (pI values of 6.2 and 5.8) with similar molecular weights (approx. 14600). Antibodies prepared against these peptides precipitate newly synthesized and secreted alpha-lactalbumin from organ cultures of mid-pregnancy mammary glands. The antibody is specific for mouse alpha-lactalbumin as it does not react with mouse casein, mouse serum or purified bovine alpha-lactalbumin or galactosyl transferase. In addition, it blocks enzymatic activity of alpha-lactalbumin in mouse milk but has no effect on guinea pig or human milk. A very sensitive radioimmunoassay has been developed with this antibody which can detect alpha-lactalbumin levels as low as 0.25 ng.

Prolactin: the forgotten hormone of human breast cancer.

Prolactin (PRL) is both a mitogen and a differentiating agent in the mammary gland. It has been shown to be involved in mammary cancer development in rodents, but in human breast cancer, its role has long been overlooked. Three criteria are applied to demonstrate PRL's involvement in this disease: (1) PRL receptors are present in human breast cancer cells, (2) human breast cancer cells in culture respond to PRL as a mitogen, and (3) PRL is synthesized by human breast cancer cells and inhibition of the binding of PRL to its receptors inhibits cell growth.

Transduction of prolactin's (PRL) growth signal through both long and short forms of the PRL receptor.

The genes for the long form of the human and the short form of the mouse PRL receptors were transfected independently into NIH 3T3 cells. Reverse transcriptase-polymerase chain reaction indicated that the transfectant designated LFH contained message for only the long form and the transfectant designated SFM had message for only the short form of the receptor. Both transfectant cell lines specifically bound lactogenic hormones with high affinity and responded to PRL in culture with a 2- to 3-fold increase in cell number preceded by transient activation of mitogen-activated protein kinase. After a PRL-responsive casein-chloramphenicol acetyl transferase (CAT) construct was introduced into both LFH and SFM cells, CAT activity was induced by PRL only in the LFH-CAT cells. Thus, while the long form of the receptor can transduce the differentiation signal, both the long and the short forms of the receptor can signal the cells to grow.

Transcriptional regulation of vascular endothelial growth factor expression in epithelial and stromal cells during mouse mammary gland development.

Accompanying changes in the development and function of the mammary gland is the establishment of a vascular network of critical importance for lactogenesis and tumorigenesis. A potent angiogenic and permeability factor that regulates vascular development in association with epithelial-stromal interactions is vascular endothelial growth factor (VEGF). Analysis of VEGF transcription by RT-PCR revealed mRNA for all three VEGF isoforms (VEGF120, 164, 188) within the mammary gland of nulliparous females. During pregnancy the level of VEGF188 declined and became undetectable during lactation in association with the increased abundance of VEGF120 and VEGF164 mRNAS: All three isoforms were expressed at consistent levels within the cleared mammary fat pad throughout development. Furthermore, the presence of VEGF188 mRNA in omental adipose tissue at various stages established that VEGF188 is expressed specifically in adipose tissue within the mammary gland. Using 3T3-L1 preadipocytes it was demonstrated that VEGF188 mRNA transcription occurs as a late event during lipogenesis distinct from earlier induction of VEGF120 and VEGF164 mRNA during differentiation. In contrast, HC11 mammary epithelial cells only expressed mRNA for VEGF120 and VEGF164. Localization of VEGF mRNA and protein revealed that VEGF is expressed in stromal cells of the mammary gland in nulliparous females and then undergoes a transition to epithelial expression during lactation. By contrast, mRNA for the VEGF receptors, Flk-1 and Flt-1, localized to stromal cells within the mammary fat pad during virgin and gestational development and was expressed in the interstitial tissue basal to epithelial cells during lactation. Taken together, these results support the conclusion that VEGF is differentially transcribed by specific cell types within the mammary gland, and that under hormonal regulation it functions in an autocrine/paracrine manner.