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Cutaneous fatty acid–binding protein (C-FABP), a cancer promoter and metastasis inducer, is overexpressed in the majority of prostatic carcinomas. Investigation of molecular mechanisms involved in tumor-promoting activity of C-FABP has established that there is a fatty acid–initiated signaling pathway leading to malignant progression of prostatic cancer cells. Increased C-FABPexpression plays an important role in this novel signaling pathway. Thus, when C-FABP expression is increased, excessive amounts of fatty acids are transported into the nucleus where they act as signaling molecules to stimulate their nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR?). The activated PPAR? then modulates the expression of its downstream target regulatory genes, which eventually lead to enhanced tumor expansion and aggressiveness caused by an overgrowth of cells with reduced apoptosis and an increased angiogenesis.

Cell migration requires the initial formation of cell protrusions, lamellipodia and/or filopodia, the attachment of the leading lamella to extracellular cues and the formation and efficient recycling of focal contacts at the leading edge. The small calcium binding EF-hand protein S100A4 has been shown to promote cell motility but the direct molecular mechanisms responsible remain to be elucidated. In this work, we provide new evidences indicating that elevated levels of S100A4 affect the stability of filopodia and prevent the maturation of focal complexes. Increasing the levels of S100A4 in a rat mammary benign tumor derived cell line results in acquired cellular migration on the wound healing scratch assay. At the cellular levels, we found that high levels of S100A4 induce the formation of many nascent filopodia, but that only a very small and limited number of those can stably adhere and mature, as opposed to control cells, which generate fewer protrusions but are able to maintain these into more mature projections. This observation was paralleled by the fact that S100A4 overexpressing cells were unable to establish stable focal adhesions. Using different truncated forms of the S100A4 proteins that are unable to bind to myosin IIA, our data suggests that this newly identified functions of S100A4 is myosin-dependent, providing new understanding on the regulatory functions of S100A4 on cellular migration.

Two subgroups of invasive breast carcinomas have been identified with a poor prognosis in different patient cohorts: the basal-like category and the subgroup containing proteins capable of inducing metastasis in experimental rodents, the metastasis-inducing proteins (MIPs). Here we identify by immunohistochemical staining for cytokeratin CK5/6 or CK14 the basal-like subgroup in a set of 297 primary invasive breast carcinomas in which the staining profile for the MIPs S100A4, osteopontin, anterior gradient-2, and S100P has already been established. Monoclonal antibodies to CK5/6 or CK14 specifically stain 31% to 34% of the primary carcinomas. These positively stained tumors are highly significantly associated with premature death of the patient (Wilcoxon statistics, P< 0.0001), the increased relative risk being approximately 5.6-fold. Positive staining for either cytokeratin is very significantly associated with that for each of the four MIPs separately and with loss of staining for the Fanconi anemia protein FANCD2 (corrected Fisher's exact test, P< 0.0007). There is no significant correlation with the remaining tumor variables tested, including staining for the estrogen receptor α, progesterone receptor, and c-erbB-2. These results show that the basal cytokeratin-like carcinomas contain many of the MIPs and that these may arise by their selection for tumors with an inherent deficiency in the FANC/BRCA pathway of DNA repair.

FANCD2, a pivotal protein in the Fanconi anemia and BRCA pathway/network, is monoubiquitylated in the nucleus in response to DNA damage. This study examines the subcellular location and relationship with prognostic factors and patient survival of FANCD2 in breast cancer. Antibodies to FANCD2 were used to immunocytochemically stain 16 benign and 20 malignant breast specimens as well as 314 primary breast carcinomas to assess its association with subcellular compartment and prognostic factors using Fisher’s Exact test or with patient survival over 20 years using Wilcoxon-Gehan statistics. Immunoreactive FANCD2 was found in the nucleus and cytoplasm of all 16 benign tissues, but nuclear staining was lost from a significant 19/20 malignant carcinomas (P< 0.0001). Antibodies to FANCD2 stained the cytoplasm of 196 primary carcinomas, leaving 118 as negatively stained. Negative cytoplasmic staining was significantly associated with positive staining for the metastasis-inducing proteins S100A4, S100P, osteopontin, and AGR2 (P≤ 0.002). Survival of patients with FANCD2-negative carcinomas was significantly worse (P< 0.0001) than those with positively stained carcinomas, and only 4% were alive at the census date. Multivariate regression analysis identified negative staining for cytoplasmic FANCD2 as the most significant indicator of patient death (P= 0.001). Thus FANCD2’s cytoplasmic loss in the primary carcinomas may allow the selection of cells overexpressing proteins that can induce metastases before surgery.

The secreted metastasis-inducing protein, human anterior gradient 2 (AGR2), has been independently reported to be associated with either a reduced or an increased survival of different groups of patients with breast cancer. We now aim to analyze the expression of AGR2 in a third completely independent group of patients using a specific AGR2 monoclonal antibody (mAb). Primary tumors from a group of 315 patients suffering from operable (stage I and II) breast cancer with 20-years follow-up were immunocytochemically stained with a specific mAb to AGR2 and associations with prognostic factors and patient survival were analyzed. The mAb specifically recognized AGR2 in Western blots, and positive staining for AGR2 was significantly associated with involved lymph nodes and staining for estrogen receptor α, progesterone receptor, and the metastasis-inducing proteins osteopontin, S100P, and S100A4. After 20 years of follow-up, only 26% of patients with AGR2-positive carcinomas survived compared with 96% of those with AGR2 negative carcinomas, with the highly significant difference in median survival times of 68 and 216 months, respectively (P< 0.0001). Cox’s multivariate regression analysis showed that staining for AGR2 was one of the most significant independent prognostic indicators, with a corrected relative risk of 9.4. The presence of AGR2 in the primary tumor is therefore a possible prognostic indicator of poor patient outcome in breast cancer.

With the widespread use of yeast two-hybrid systems, many heterodimeric forms of S100 proteins have been found, although their biological significance is unknown. In the present study, S100A1 was found to interact with another S100 protein, S100P, by using the yeast two-hybrid system. The binding parameters of the interaction were obtained using an optical biosensor and show that S100P has a slightly higher affinity for S100A1 (Kd=10–20 nM) when compared with that for self-association (Kd=40–120 nM). The physical interaction of S100A1 and S100P was also demonstrated in living mammalian cells using a fluorescence resonance energy transfer technique. Preincubation of recombinant S100P with S100A1, before the biosensor assay, reduced by up to 50% the binding of S100P to a recombinant C-terminal fragment of non-muscle myosin A, one of its target molecules. Site-specific mutations of S100P and S100A1, combined with homology modelling of an S100P/S100A1 heterodimer using known S100P and S100A1 structures, allowed the hydrophobic interactions at the dimeric interface of the heterodimer to be defined and provide an explanation for the heterodimerization of S100P and S100A1 at the molecular level. These results have revealed the similarities and the differences between the S100P homodimer and the S100A1/S100P heterodimer.

We examine the relationship between the chain length of heparin-derived oligosaccharides, fibroblast growth factor (FGF)-2 binding kinetics and the ability of the oligosaccharides to allow FGF-2-induced proliferation of chlorate-treated rat mammary fibroblasts. First, using an optical biosensor, we show that FGF-2 did not bind disaccharides, but definitively bound to tetrasaccharides. As the chain length increased from tetrasaccharide to octasaccharide, there was a substantial increase in kass (564000M−1·s−1 to 2000000M−1·s−1, respectively) and affinity (Kd 77nM to 11nM, respectively) for FGF-2. From decasaccharides and longer, the kass and affinity for FGF-2 was reduced substantially (tetradecasaccharide kass 470000M−1·s−1, Kd 30nM). In chlorate-treated, and hence sulphated, glycosaminoglycan-deficient cells, FGF-2 alone or in the presence of disaccharides did not stimulate DNA synthesis and it only elicited an early transient dual phosphorylation of p42/44 mitogen-activated protein kinase (MAPK). In the same cells FGF-2 in the presence of tetrasaccharides and longer oligosaccharides was able to restore DNA synthesis and enable the sustained dual phosphorylation of p42/44MAPK. However, the oligosaccharides from tetrasaccharides to octasaccharides were less potent in proliferation assays than deca- and longer oligosaccharides. Therefore, there was no correlation between the binding parameters and the potency of the oligosaccharides in DNA synthesis assays. These results demonstrate that tetrasaccharides are able to bind FGF-2 and enable FGF-2 to stimulate cell proliferation, which sets important boundary conditions for models of the FGF-2—heparan sulphate—FGF receptor complex.

Muc4 (also called sialomucin complex), the rat homolog of human MUC4, is a heterodimeric glycoprotein complex that consists of a peripheral O-glycosylated mucin subunit, ASGP-1, tightly but noncovalently linked to a N-glycosylated transmembrane subunit, ASGP-2. The complex is expressed in a number of normal, vulnerable epithelial tissues, including mammary gland, uterus, colon, cornea and trachea. Muc4/SMC is also overexpressed or aberrantly expressed on a number of human tumors including breast tumors. Overexpression of Muc4/SMC has been shown to block cell-cell and cell-matrix interactions, protect tumor cells from immune surveillance and promote metastasis. In addition, as a ligand for ErbB2, Muc4/SMC can potentiate phosphorylation of ErbB2 and potentially alter signals generated from this receptor. Using A375 human melanoma cells and MCF7 human breast adenocarcinoma cells stably transfected with tetracycline regulatable Muc4, we have investigated whether overexpression of Muc4/SMC can repress antibody binding to cell surface-expressed ErbB2. Overexpression of Muc4/SMC does not affect the level of ErbB2 expression in either cell line, but it does reduce binding of a number of anti-ErbB2 antibodies, including Herceptin. Interestingly, overexpression of ErbB2 does not block binding of other unrelated antibodies of the same isotype, suggesting that the reduction in ErbB2 antibody binding is due to complex formation of Muc4/SMC and ErbB2. Furthermore, capping of Muc4/SMC with anti-Muc4/SMC antibodies reduces antibody binding to ErbB2 instead of increasing binding, again suggesting that reduced antibody binding to ErbB2 is due to steric hindrance from complex formation of Muc4/SMC and ErbB2. Thus, overexpression of Muc4/SMC on tumor cells may have both prognostic and therapeutic relevance. © 2002 Wiley-Liss, Inc.

The calcium‐binding protein S100A4 induces the metastatic phenotype in rodent models of breast cancer and its expression correlates strongly with reduced survival in human breast cancer. The expression of S100A4 in normal bladders and 101 bladder tumours has been studied using immunocytochemistry. Moderate or strong expression of S100A4 was found in 28% of the tumours, whilst the remaining tumours and normal urothelium either failed to stain or showed weak staining. S100A4 staining was more frequently observed in invasive bladder tumours than in non‐invasive tumours (p<0.05). In invasive tumours, S100A4 staining was usually strongest in invasive regions and single infiltrating cells. Statistically significant associations were found between S100A4 expression and metastasis (p=0.0003) and reduced survival (p<0.0001). It is concluded that S100A4 expression may play an important role in bladder cancer and may identify a subgroup of patients at increased risk of metastasis who should be considered for adjuvant systemic therapy. Copyright © 2002 John Wiley & Sons, Ltd.

The calcium-binding protein S100A4 induces the metastatic phenotype in rodent models of breast cancer, and its expression strongly correlates with reduced survival in human breast and bladder cancer. We have established an orthotopic model of bladder cancer by injecting a cell line derived from a carcinogen-induced rat bladder tumor into the muscular wall of syngeneic rats. MYU-3L cells produce rapidly growing, invasive tumors in the bladder wall but they fail to metastasize. Transfection of MYU-3L cells with a plasmid vector directing overexpression of the S100A4 gene generates variants in which S100A4 expression is elevated by up to sevenfold in comparison with the untransfected cells. Variants overexpressing S100A4 produce primary tumors at similar frequencies and latencies to the parental cell line, a significant number of which metastasize to the para-aortic lymph nodes or lungs. Expression of S100A4 protein in the primary tumors was heterogeneous, but was stronger and more consistent in the metastases, suggesting that transfectants overexpressing S100A4 possess an enhanced ability to form metastatic lesions. We conclude that overexpression of S100A4 can induce the metastatic phenotype in this rodent model of bladder cancer. Taken together with the results from our parallel studies of human bladder cancer, these data suggest a significant role for S100A4 in bladder cancer metastasis and identify a potential new target for systemic therapy in patients with this disease.

Experimentally elevated levels of S100A4 induce a metastatic phenotype in benign mammary tumour cells in vivo. In humans, the presence of S100A4 in breast cancer cells correlates strongly with reduced patient survival. Potential interacting binding partners for S100A4 have now been examined using an optical biosensor. There was significant interaction of S100A4 with non-muscle myosin and p53, but not with actin, tropomyosin or tubulin. The results suggest that myosin and p53 are likely to be intracellular targets of S100A4. S100A4 had a greater affinity for wild-type or mutant arg-175-his p53 than for non-muscle myosin. The results suggest that S100A4 might induce metastasis by influencing the function of p53 as well as through its interaction with myosin and that any mechanism is independent of the mutational status of p53.

Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycoprotein composed of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. SMC/Muc4 is highly expressed on the surface of 13762 rat mammary adenocarcinoma cells at approximately 100 times the level found in the lactating mammary gland. Immunocytochemical staining of SMC/Muc4 in the developing rat mammary gland is localized to the apical membrane of the ductal epithelium. This staining pattern is similar to that for peanut lectin, a differentiation marker, which binds to cells expressing the disaccharide Thomsen–Friedenreich or TF antigen. Blotting of glycoproteins expressing the TF antigen from mammary tissues with peanut lectin detects a protein matching the migration of ASGP-2. Analysis of immunoprecipitated SMC/Muc4 by peanut lectin blotting shows that the TF antigen is abundantly present on the ASGP-2 subunit, hence the similarity of staining pattern with SMC/Muc4 antisera and peroxidase-conjugated lectin in mammary tissues. The TF antigen is also present on ASGP-2 of SMC/Muc4 produced by confluent cultures of Rama 37 rat mammary epithelial stem cells after their induction to an alveolar-like phenotype with prolactin. These results indicate that the TF antigen is present on the ASGP-2 transmembrane subunit of SMC/Muc4 from phenotypically normal tissues and cells, in contrast to malignant cells whose peanut lectin-binding disaccharide is located on ASGP-1. © 2001 Wiley-Liss, Inc.

Fibroblast growth factor-2 (FGF-2) interacts with a dual receptor system consisting of tyrosine kinase receptors and heparan sulfate proteoglycans (HSPGs). In rat mammary fibroblasts, FGF-2 stimulated DNA synthesis and induced a sustained phosphorylation of p42/44MAPKand of its downstream target, p90RSK. Moreover, FGF-2 also stimulated the transient degradation of IκBα and IκBβ. PD098059, a specific inhibitor of p42/44MAPKphosphorylation, inhibited FGF-2-stimulated DNA synthesis, phosphorylation of p42/44MAPKand p90RSK, and degradation of IκBβ. In contrast, in chlorate-treated and hence sulfated glycosaminoglycan-deficient cells, FGF-2 was unable to stimulate DNA synthesis. However, FGF-2 was able to trigger a transient phosphorylation of both p42/44MAPKand p90RSK, which peaked at 15 min and returned to control levels at 30 min. In these sulfated glycosaminoglycan-deficient cells, no degradation of IκBα and IκBβ was observed after FGF-2 addition. However, in chlorate-treated cells, the addition of heparin or purified HSPGs simultaneously with FGF-2 restored DNA synthesis, the sustained phosphorylation of p42/44MAPKand p90RSK, and the degradation of IκBα and IκBβ. These results suggest that the HSPG receptor for FGF-2 not only influences the outcome of FGF-2 signaling, e.g.cell proliferation, but importantly regulates the immediate-early signals generated by this growth factor.

A panel of human breast cancer specimens was examined for single base change mutations by DNA sequencing and for larger deletions using a PCR-based assay. In the cancer specimens examined, no sequencing variants were detected other than a previously characterized polymorphism. Although most of the specimens contained estrogen receptor (ER) variants at a low level, 2 of 118 specimens exhibited variants which, after amplification, constituted most of the amplified ER cDNA. One specimen contained a single variant form, and there was little evidence of the wild-type ER mRNA by PCR, Northern blotting or immunocytochemistry. The second specimen, despite the presence of a normal-sized mRNA by Northern blotting and normal immunocytochemical staining for ER, contained at least 5 different variant forms as well as the wild-type ER. All but 1 of the variant forms were processing variants, and 3 of these processing variants have not been described before. One variant, although lacking exons 2–4, has break points in exons 1 and 5 that do not correspond to intron–exon boundaries. This variant might reflect more widespread damage to the genome in this breast cancer specimen. The low level of occurrence of variants suggests that ER variant forms, at least in the coding region, do not contribute generally to the progression of breast cancer. Int. J. Cancer 88:209–216, 2000. © 2000 Wiley-Liss, Inc.

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparan/dermatan sulfate-binding growth factor produced by stromal cells that acts as a paracrine effector on neighboring epithelia. HGF/SF stimulated DNA synthesis in human mammary (Huma) 109 myoepithelial-like cells grown on collagen I and fibronectin substrata but not when grown on plastic. Dual phosphorylation of mitogen-activated protein kinases (p42/44MAPK) was required for this stimulation of DNA synthesis. In Huma 109 cells cultured on plastic, HGF/SF stimulated a transient phosphorylation of p42/44MAPK, which reached a maximum at 10 min after addition of the growth factor and returned to near basal levels after 20 min. In contrast, the phosphorylation of p42/44MAPKstimulated by HGF/SF in cells cultured on collagen I or fibronectin was sustained over 45 min. In Huma 109 cells deficient in sulfated glycosaminoglycans, HGF/SF failed to stimulate p42/44MAPKphosphorylation or DNA synthesis on any substratum, even when soluble heparan sulfate proteoglycans purified from the cells or from the culture medium were added. However, HGF/SF stimulated DNA synthesis and a sustained phosphorylation of p42/44MAPKin sulfated glycosaminoglycan-deficient Huma 109 cells plated on a substratum of medium HSPGs but not cell HSPGs. The HGF/SF-induced proliferation is thus highly dependent on heparan sulfate proteoglycans in myoepithelial-like cells.

The calcium-binding protein S100A4 (p9Ka) has been shown to cause a metastatic phenotype in rodent mammary tumor cells and in transgenic mouse model systems. mRNA for S100A4 (p9Ka) is present at a generally higher level in breast carcinoma than in benign breast tumor specimens, and the presence of immunocytochemically detected S100A4 correlates strongly with a poor prognosis for breast cancer patients. Recombinant S100A4 (p9Ka) has been reported to interact in vitrowith cytoskeletal components and to form oligomers, particularly homodimers in vitro. Using the yeast two-hybrid system, a strong interaction between S100A4 (p9Ka) and another S100 protein, S100A1, was detected. Site-directed mutagenesis of conserved amino acid residues involved in the dimerization of S100 proteins abolished the interactions. The interaction between S100A4 and S100A1 was also observed in vitrousing affinity column chromatography and gel overlay techniques. Both S100A1 and S100A4 can occur in the same cultured mammary cells, suggesting that in cells containing both proteins, S100A1 might modulate the metastasis-inducing capability of S100A4.

Rodent S100A4 (p9Ka) induces a metastatic phenotype in benign rat mammary tumour cells and cooperates with the neu oncogene to produce metastatic tumours in a transgenic mouse model system. Human S100A4 possesses similar metastasis-inducing properties. S100A4 mRNA is now sought in human breast tumour-derived cell lines and tumour specimens. S100A4 mRNA is present in some cell lines derived from malignant breast cancers, but is not detectable in cells derived from benign breast tumours. In human tumour specimens, using in situ hybridisation, the mRNA for S100A4 is localised to the epithelial cells of carcinoma specimens, and in some normal breast specimens, to a stromal region surrounding the epithelial ducts. In carcinoma specimens, S100A4 mRNA is also found in the stromal region surrounding islands of cancer cells. For both the epithelial and stromal components, S100A4 mRNA is present at a higher level in carcinomas relative to benign breast tumour specimens. In general, there is a concordance between the S100A4 mRNA signal from the epithelial and stromal elements of the same carcinoma specimens. Using Northern blotting techniques, these results have been extended to a panel of 137 benign and malignant breast tumour specimens. The results show that S100A4 mRNA occurs in the more-malignant, rather than in the more-benign tumour specimens. Int. J. Cancer 86:219–228, 2000. © 2000 Wiley-Liss, Inc.

The calcium-binding protein S100A4 (p9Ka) has been shown to cause a metastatic phenotype in rodent mammary tumor cells and in transgenic mouse model systems. mRNA for S100A4 (p9Ka) is present at a generally higher level in breast carcinoma than in benign breast tumor specimens, and the presence of immunocytochemically detected S100A4 correlates strongly with a poor prognosis for breast cancer patients. Recombinant S100A4 (p9Ka) has been reported to interact in vitro with cytoskeletal components and to form oligomers, particularly homodimers in vitro. Using the yeast two-hybrid system, a strong interaction between S100A4 (p9Ka) and another S100 protein, S100A1, was detected. Site-directed mutagenesis of conserved amino acid residues involved in the dimerization of S100 proteins abolished the interactions. The interaction between S100A4 and S100A1 was also observed in vitro using affinity column chromatography and gel overlay techniques. Both S100A1 and S100A4 can occur in the same cultured mammary cells, suggesting that in cells containing both proteins, S100A1 might modulate the metastasis-inducing capability of S100A4.

Prostaglandin F2α (PGF2α), a mitogen for Swiss 3T3 cells, triggers cyclin D1 mRNA/protein expression prior to cellular entry into the S phase, but fails to raise cdk4 or cyclin D3 levels, while 1-oleoyl-2-diacylglycerol (OAG), a protein kinase C (PKC) and tyrosine kinase (TK) activator, induces only cyclin D1 expression with no mitogenic response. In contrast, in PKC-depleted or -inhibited cells, PGF2α, but not OAG, increases cyclin D1 expression with no mitogenic response. Finally, OAG, in the presence of orthovanadate (Na3VO4) or TGFβ1, induces DNA synthesis. Thus, it appears that PGF2α triggers cyclin D1 expression via two independent signaling events that complement with TGFβ1-triggered events to induce DNA synthesis.

1Unilateral changes in mammary cell number are elicited when one gland is milked more or less frequently than the contralateral gland in lactating goats. These changes were investigated using histochemical and immunocytochemical markers of mammary cell types, and the degree of mammary apoptosis was determined by end‐labelling of fragmented DNA.2Histological analysis confirmed that unilateral cessation of milking initiated involution and cell loss preferentially in the unmilked gland. The presence of fragmented DNA and morphological characteristics consistent with apoptosis demonstrated that these changes in mammary cell number in unmilked glands were, in part, the result of programmed alveolar cell death.3De‐differentiation of the remaining secretory cells to ductal epithelial cells occurred with an increase in staining of cytokeratin markers and decreased staining by peanut lectin and casein antisera.4Differential once‐ and thrice‐daily milking of lactating goats was also associated with unilateral changes in mammary cell number and milk yield. Milk yield and alveolar size were reduced after 4 weeks of infrequent milking. The latter was due to the increased loss of secretory cells by apoptosis, as indicated by a higher degree of fragmented DNA laddering.5After 10 weeks of differential milking, a homogeneous secretory morphology, albeit with smaller alveoli, was maintained in thrice‐daily milked glands. Once‐daily milked glands possessed a heterogeneous composition of terminal structures, resulting in the simultaneous presence of secretory and involuting alveoli as well as resting ductules.6The differences in programmed cell death and mammary morphology between unmilked and twice‐daily milked glands, and between once‐ and thrice‐daily milked glands, suggests that mammary apoptosis is subject to modulation by intra‐mammary mechanisms sensitive to the frequency of milk removal.

Human cDNAs corresponding to two epidermal growth factor-related products that are overexpressed in human breast cancers, that for c-erbB-2 (HER-2) and for transforming growth factor α (TGFα), have been cloned downstream of the mouse mammary tumor virus (MMTV) long terminal repeat promoter and injected into the pronucleus of fertilized oocytes of Sprague-Dawley rats to produce transgenic offspring. Expression of the transgenic mRNAs is not detectable in mammary tissue from virgin transgenic rats but is detected in mammary tissue from certain lines of mid-pregnant transgenic rats. When two such lines of either type of transgenic rat are subjected to repeated cycles of pregnancy and lactation, they produce, primarily in the mammary glands, extensive pathologies, whereas virgin transgenic rats produce no such abnormalities. Multiparous transgenic female offspring from c-erbB-2-expressing lines develop a variety of focal hyperplastic and benign lesions that resemble lesions commonly found in human breasts. These lesions include lobular and ductal hyperplasia, fibroadenoma, cystic expansions, and papillary adenomas. More malignant lesions, including ductal carcinoma in situand carcinoma, also develop stochastically at low frequency. The mammary glands of transgenic females invariably fail to involute fully after lactation. Similar phenotypes are observed in female MMTV-TGFα transgenic rats. In addition, multiparous TGFα-expressing female transgenics frequently develop severe pregnancy-dependent lactating hyperplasias as well as residual lobules of hyperplastic secretory epithelium and genuine lactating adenomas after weaning. These transgenic rat models confirm the conclusions reached in transgenic mice that overexpression of the c-erbB-2 and TGFα genes predisposes the mammary gland to stochastic tumor development.

The family of S-100-related proteins consists of a number of small potential calcium-binding proteins of unknown function. Elevated expression of one of these proteins, p9Ka, or of its mRNA, correlates with the metastatic potential of cultured mammary epithelial cells from rat or mouse. Over-expression of p9Ka by transfection of benign rat mammary epithelial tumor cells with the gene for p9Ka induces the metastatic phenotype. At present there is little information on the occurrence of p9Ka in normal rat tissues. A specific antiserum immunocytochemically detects p9Ka intracellularly in most normal adult rat tissues studied, including smooth muscle, brown adipose tissue, and liver. In other tissues, p9Ka is localized specifically to some absorptive and keratinized epithelia, the acid-secreting parietal cells of the stomach, the neuronal cells within plexuses of the autonomic nervous system, and a proportion of cells of the immune system in spleen, lymph nodes, bone marrow, and blood. p9Ka is found widely in both arteries and veins, particularly in the smooth muscle and in the endothelium of smaller veins. In mammary gland, the pattern of staining suggests that p9Ka is extracellularly located in a region surrounding the ducts.

Elevated intracellular levels of S100A4, an S100-related calcium-binding protein, induce metastatic capability in benign mammary tumor-derived epithelial cells and in transgenic mice bearing oncogene-induced benign mammary tumors. The S100A4(p9Ka) gene in rat mammary epithelial cells expressing low levels of S100A4 yields a reduced number of fragments upon digestion with the methylation-sensitive restriction enzyme, HpaII, compared with the gene from high S100A4-expressing cells. Genomic sequencing of two potential regulatory elements in the S100A4 gene, an intronic enhancer and TATA box region, revealed that in low S100A4-expressing cells, most cytosine bases exhibited high levels of resistance to conversion to thymine by sodium bisulfite. In derivative cell lines, which express high levels of S100A4, only a small number of cytosine bases were resistant to treatment with sodium bisulfite. In contrast, cytosine bases in the DNA surrounding an upstream regulatory region, which binds inhibitory GC factor in the low-expressing cell lines, are sensitive to conversion to thymine by sodium bisulfite in both low- and high-expressing cell lines. The results suggest that the rat S100A4 gene is maintained in a different state in the low-expressing cell lines and that this state might be a consequence of the pattern of methylation in this regulated gene that does not contain a CpG island.

Maximal stimulation of DNA synthesis in quiescent rat mammary (Rama) 27 fibroblasts is elicited by epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) 18 h after the initial addition of the growth factors-the ‘lag’ period. At maximally-stimulating concentrations, EGF and bFGF are interchangeable 9 h after their initial addition. When the initial concentration of growth factor is below that required to elicit a maximal response, it is possible to increase the level of DNA synthesis by increasing the concentration of growth factor 9 h after its initial addition. When the initial concentration of growth factor is high, substitution by a lower concentration of growth factor after 9 h allows a greater proportion of cells to synthesize DNA than would be expected from a continuous low dose of growth factor. Similar results are obtained when both the growth factor and its concentration are changed 9 h after the initial addition of growth factor. However, when EGF at a low concentration is substituted for a high concentration of EGF or bFGF the resulting increase in the levels of DNA synthesis is greater when EGF rather than bFGF is added for a second time. The half-life of the growth-stimulatory signals delivered by EGF and by bFGF 9 h after their initial addition is 1–2 h. These results suggest that to stimulate DNA synthesis: (i) EGF or bFGF must deliver a signal(s) continuously; (ii) the initial signals produced by EGF and bFGF are equivalent; (iii) the signals produced between 9–18 h by EGF may be different to those produced by bFGF.

Maximal stimulation of DNA synthesis in quiescent rat mammary (Rama) 27 fibroblasts is elicited by epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) 18 h after the initial addition of the growth factors-the ‘lag’ period. At maximally-stimulating concentrations, EGF and bFGF are interchangeable 9 h after their initial addition. When the initial concentration of growth factor is below that required to elicit a maximal response, it is possible to increase the level of DNA synthesis by increasing the concentration of growth factor 9 h after its initial addition. When the initial concentration of growth factor is high, substitution by a lower concentration of growth factor after 9 h allows a greater proportion of cells to synthesize DNA than would be expected from a continuous low dose of growth factor. Similar results are obtained when both the growth factor and its concentration are changed 9 h after the initial addition of growth factor. However, when EGF at a low concentration is substituted for a high concentration of EGF or bFGF the resulting increase in the levels of DNA synthesis is greater when EGF rather than bFGF is added for a second time. The half-life of the growth-stimulatory signals delivered by EGF and by bFGF 9 h after their initial addition is 1–2 h. These results suggest that to stimulate DNA synthesis: (i) EGF or bFGF must deliver a signal(s) continuously; (ii) the initial signals produced by EGF and bFGF are equivalent; (iii) the signals produced between 9–18 h by EGF may be different to those produced by bFGF.

The S100-related calcium-binding protein S100A4 (p9Ka) is expressed at a low level in rat mammary epithelial cells from normal mammary gland and benign mammary tumors. In transgenic mice, expressed rat S100A4 transgenes co-operate with the activated c-erbB-2 oncogene, neu, to form metastatic mammary tumors. Elevated levels of S100A4 (p9Ka) in cultured benign rat or mouse mammary epithelial cells are associated with the induction of metastatic capability. A cis-acting sequence related to the consensus recognition sequence of GC-factor, 1,300 base pairs upstream of the start site of transcription of the rat S100A4 gene, acts as a cis-acting inhibitor of transcription of the S100A4 (p9Ka) gene in a low S100A4 (p9Ka)-expressing benign rat mammary epithelial cell line, but not in highly expressing rat mammary epithelial cell lines. There is an inverse relationship between the level of S100A4 (p9Ka) mRNA and the level of GC-factor mRNA in a range of rat mammary cell lines. The results suggest a novel mechanism for regulating the expression of the mRNA encoding an S100 protein.

We raised antiserum to human recombinant basic fibroblast growth factor (rbFGF) in rabbits. With this affinity-purified antiserum, other antisera to rbFGF, and commercial antiserum to bovine pituitary bFGF, we undertook immunocytochemical detection of bFGF in histological sections of rat mammary glands at different developmental stages. In non-growing ducts, anti-bFGF serum stains the basement membrane/myoepithelial cells, whereas in serial sections most of this stain is observed to be associated with anti-laminin-staining basement membranes rather than with anti-callus-keratin-staining myoepithelial cells. The weak staining of the myoepithelial cells is enhanced when NiCl2 is included in the detection system, but little staining for bFGF is observed in the epithelial cells. In growing neonatal ducts from 1-day-old rats, in growing terminal end buds (TEBs) and, to a lesser extent, in growing alveolar buds (ABs) in prepubescent (21-day) and pubescent (50-day) rats, both their inner and outer cells are stained moderately by anti-bFGF sera. In non-growing ducts from rats aged 6 days, in non-growing ABs of rats aged 60 days and more, and in alveoli from pregnant and lactating rats, only the basement membrane/myoepithelial cell area is stained by anti-bFGF sera; the epithelial cells are unstained. Staining of the myoepithelial cells is enhanced by mixtures of rbFGF and anti-bFGF sera in non-growing ducts, but there is little change in the staining of growing TEBs. All staining by anti-bFGF sera is abolished with heparin in the reactions. We suggest that the immunoreactive bFGF is present mainly bound to heparan sulfate glycosaminoglycans in the basement membrane of resting structures, but that immunoreactive bFGF becomes associated with proliferating cells, particularly those intermediate in characteristics between epithelial and myoepithelial cells in growing structures such as TEBs.

Peroxidase-conjugated Griffonia simplicifolia-1 (GS-1) and pokeweed mitogen (PWM) histochemically stain only the myoepithelial cells and not the epithelial or fibroblastic cells of rat mammary glands preserved in methacarn or glutaraldehyde and embedded in paraffin. This pattern of staining occurs in other rat exocrine glands except the pancreas, but is the reverse of that seen in most lining epithelium. The histochemical binding of GS-1 and PWM to myoepithelial cells is inhibited specifically by D-galactose and by polymers of N-acetylglucosamine, respectively. GS-1 and its subcomponent, GS-1-B4, also bind to extracellular structures similar to those stained by anti-laminin serum. At the ultrastructural level, both conjugated GS-1 and PWM bind to the plasma membrane of the myoepithelial cells, as well as to the adjacent basement membrane. Non-metastasizing rat mammary tumors produced by dimethylbenz[a]anthracene, by derivative epithelial stem-cell lines, and by a transplantable tumor all contain more elongated myoepithelium-like cells as well as cuboidal epithelium-like cells; both cell types are neoplastic. The more elongated myoepithelium-like cells are stained by GS-1 and PWM, whereas the cuboidal epithelium-like cells are unstained. Moderately and strongly metastatic rat mammary tumors produced by epithelial cell lines and by transplantable tumors, respectively, contain no such neoplastic cells that bind either lectin. We suggest that the carbohydrate receptors for GS-1 and PWM are consistent markers for the presence of the myoepithelial cell in normal and tumorous rat mammary glands.

The histochemical binding of peroxidase-conjugated Griffonia simplicifolia-1 (GS-1) and pokeweed mitogen (PWM) to methacarn-preserved and paraffin-embedded female rat mammary glands at different developmental stages has been undertaken with a view to investigating the ontogeny of the myoepithelial cell. Conjugated GS-1 fails to stain the outer layer of ductal cells in neonatal rats up to 3 days old, but thereafter the staining increases so that all such cells are intensely stained in rats 5 days old and in mature, pregnant, and lactating rats. Conjugated GS-1 also stains most of the inner epithelial cells that line the ducts in neonatal rats up to about 5 days after birth; thereafter no such cells are stained in mature, pregnant, and lactating rats. Conjugated PWM stains both the inner and outer cell layers of ducts in neonatal rats up to 5 days old; thereafter the fraction of strongly stained cells declines rapidly in both cell layers so that in 6-day-old rats only weak staining is visible. Staining with PWM continues to decline for the inner epithelial cells of the ducts until it ceases when the rats mature; in contrast, that for the outer cell layer of the ducts increases so that all such cells are stained intensely when the rats mature. This pattern of ductal staining with PWM is maintained for pregnant and lactating rats. In terminal end buds of mammary ducts of prepubertal rats, GS-1 binds mainly to the peripheral or cap cells, the staining intensity increasing from cap cells at the distal tip to myoepithelial cells of the subtending duct. PWM binds to many more of the cortical epithelial cells and fewer of the cap cells. At the ultrastructural level, cap cells and adjacent immature myoepithelial cells both bind GS-1 and PWM to their surfaces, but basal clear cells do not. In alveolar buds and in alveoli, both conjugated lectins GS-1 and PWM bind to myoepithelial cells but not to epithelial cells of the rat mammary gland. We suggest that the appearance of carbohydrate receptors for GS-1 and PWM marks specific stages of myoepithelial cell differentiation in developing rat mammary glands.

Antiserum to epithelial membrane antigen and three monoclonal antibodies (MAb) to milk-fat globule membranes immunocytochemically stain only epithelial cells, whereas a fourth reacts also with myoepithelial cells in inter- and intralobular ducts of human breast. Staining with peanut lectin shows a gradual increase for epithelial cells, from little or no staining in ducts through variable staining in ductules to intense staining in secretory alveoli. Antisera and MAb to vimentin, smooth-muscle actin, MAb to the common acute lymphoblastic leukemia antigen and to a glycoprotein of 135 KD stain myoepithelial cells in main ducts, but this staining is reduced in inter- and intralobular ducts and ductules. MAb to epithelial-specific keratin 18 stain a minor population of ductal epithelial cells, the major population of epithelial cells in interlobular (ILD) and extralobular terminal ducts (ETD), and epithelial cells in a minority of ductules. In lactating glands most epithelial cells in ductules are stained, but the alveolar and myoepithelial cells are unstained. Keratin MAb PKK2 and LP34 strongly stain myoepithelial cells, but only a minor population of epithelial cells in main ducts. However, these MAb stain principally the epithelial cells in ILD, ETD, and a minority of ductules. In lactating glands most epithelial cells are stained in ductules, but the myoepithelial and not the alveolar cells are stained intensely in secretory lobules. It is suggested that the unusual staining pattern of cells found principally in the ILD, ETD, and some ductules may represent regions of growth and/or subpopulation(s) of cells intermediate between epithelial and myoepithelial cells.

In the 100-day-old virgin and lactating rat mammary glands, type V collagen is mainly present in the interstitial connective tissue and in association with blood vessels. It is not present in the basement membrane region surrounding the ducts in mature virgin glands but is present in this region in neonatal and lactating glands. Ultrastructural localization of type V collagen reveals that it is mainly located on the basal surface (i.e., the surface in contact with the basement membrane) of epithelial but not myoepithelial cells. In addition, type V collagen is located on some interstitial collagen fibers and on a large number of granules that are in close proximity to the basal surface of both epithelial and myoepithelial cells. Immunofluorescence and biochemical studies indicate that several clonal mammary fibroblastic cell lines synthesize type V collagen in vitro. In some cell lines, type V collagen is secreted as an extensive fibrillar meshwork on the surface of the cells, whereas in other cell lines, it is secreted beneath the cells around their periphery. A number of mammary epithelial and myoepithelial-like cells, however, do not synthesize type V collagen in vitro.

Evidence based on immunocytochemical staining and ultrastructure suggests that morphological gradations between epithelial and myoepithelial cells, and possibly between epithelial cells and alveolar-like cells occur in terminal ductal structures of rat and human mammary glands. The benign carcinogen-induced rat and benign human mammary tumors can contain epithelial, myoepithelial-like and alveolar-like cells, whereas the malignant counterparts mainly contain only epithelial-like cells. Clonal epithelial cell lines from normal rat mammary glands, benign tumors, and SV40-transformed human mammary glands can differentiate to either myoepithelial-like or alveolar-like cells. In those of the rat, the differentiation processes occur in steps: intermediate cells along the myoepithelial-like pathway resemble intermediates in terminal ductal structures in vivo, and can also generate certain well-differentiated mesenchymal elements of the original tumours. Differentiation of the benign rat cells to alveolar-like cells with mammatrophic hormones and retinoids in vitro leads to a reduction in their tumor-forming ability in vivo. Cell lines from malignant rat mammary tumors of increasing metastatic potential and from human ductal carcinomas largely fail to yield myoepithelial-like or alveolar-like cells and are relatively slow-growing. Growth of the rat mammary epithelial cells in culture is stimulated by a pituitary-derived mammatrophic growth factor (PMGF), prostaglandin E2, and a-transforming growth factor; the response of the malignant cell lines to PMGF is reduced. It is suggested that stem cells exist in the rat and human glands that are capable of differentiating to the other major cell types of the mammary parenchyma, and that during the carcinogenic process they generate genetically unstable cells which lose their ability to differentiate and attempt to maximise their intrinsically slow growth rate.

CHEMICAL1or viral2transformation of fibroblasts in tissue culture results in cells with altered properties (for review see ref. 2). These properties can be broadly subdivided into those associated with changes in the plasma membrane3and those associated with intracellular regulatory changes2. The former changes include the ability to grow in soft agar4, the loss of an iodinatable surface glycoprotein5,6and an increase in total cell-membrane-associated proteolytic activity7on cell transformation. The chief manifestation of intracellular changes is the failure of transformed fibroblasts to cease growing at a specific point (G1or G0) in the cell cycle8and thus to express the typical increases in protein synthesis, ribosomal RNA synthesis and DNA synthesis upon growth reinitiation9. We have now investigated some of these properties in cell mutants of BALB/c–3T3 which show temperature-dependent changes in many properties characteristic of the transformed state in tissue culture. These mutant cells behave as normal mouse fibroblasts in the regulation of their internal cellular machinery at permissive or non-permissive temperatures but show temperature sensitive changes in those properties we have measured which are considered to be associated with membranes.

mRNA for basic Fibroblast Growth Factor (bFGF) was expressed in a series of SV40-transformed human mammary cell lines as molecules of 7.1, 3.6, 2.0 and 1.2 kb. This expression was much weaker in those lines of epithelial morphology than in myoepithelial-like cell lines derived from them. It was confirmed, using northern hybridization to single-stranded RNA probes, that the multiple mRNAs were transcribed from the coding strand for bFGF. bFGF activity was detected in extracts of the cells and the relative amounts of activity corresponded in general to the amounts of mRNA found. Similar results were obtained from spontaneously transformed cell lines derived from a human benign breast lesion. The presence of bFGF protein in the extracts was confirmed by western blotting, which showed a band of 18-19 kDa, migrating in the same position as authentic bFGF; in addition, the myoepithelial-like cells showed prominent bands of bFGF at 24 and 26 kDa. No FGF receptor was detectable by the binding of 125I-bFGF to the SV40-transformed cell lines or to the epithelial cell lines from the benign breast lesion, but both high- and low-affinity receptors were found on myoepithelial-like cells derived from the latter. The results indicate that differentiation to the human myoepithelial-like phenotype in culture is associated with the enhanced expression of bFGF, and it is suggested that bFGF, immunocytochemically detected in the basement membrane of the human breast, may arise, at least in part, from the myoepithelial cells of the mammary parenchyma.

The cell-surface receptor tyrosine kinase protein c-erbB-2 is immunocytochemically detected as membrane staining on the surface of cancer cells in 20–30% of cases of breast cancer, and its presence has been associated with poor prognosis for the patient. However, there have been numerous reports of immunocytochemical staining for c-erbB-2 solely in the cytoplasm of some normal and tumour specimens with frequently used anti-sera, and the presence of such staining has been difficult to interpret. It is not known for certain that cytoplasmic c-erbB-2 staining is an artefact of the immunocytochemical procedures used. Thus, mRNA for c-erbB-2 has been quantified in tumours exhibiting only cytoplasmic staining or varying levels of membrane staining using a sensitive, competitive PCR method. Whereas abundant levels of c-erbB-2 mRNA are found in tumours exhibiting membrane staining for c-erbB-2 and these levels correlate with the percentage of tumour cells showing membranous staining for c-erbB-2, the level of c-erbB-2 mRNA in tumours displaying only cytoplasmic staining is no higher than in c-erbB-2-negative specimens. Int. J. Cancer 76:459–463, 1998.© 1998 Wiley-Liss, Inc.

Nontransformed animal fibroblasts in tissue culture regulate total protein and ribosomal RNA synthesis coordinately with changes in the cellular growth state. Here it is shown that the amounts and rates of synthesis of cytoplasmic poly(A)-containing RNA, presumed to be mRNA, do not appreciably change with alterations in growth state. In nongrowing (resting) cultures of BALB/c 3T3 cells presumptive mRNA molecules predominantly accumulate as cytoplasmic ribonucleoprotein particles; the RNA from which can be chased into the polyribosomal structure upon activation of the resting cultures with animal sera. It is suggested that the decreased protein synthetic rate in resting as compared with growing cells is in part due to a failure of preexisting mRNAs to attach to ribosomes and that addition of serum to the tissue culture medium can partially overcome this translational lesion.

THE induction of cell growth by animal serum in quiescent cultured fibroblasts is preceded by a sequence of regulated steps1. These steps include stimulation of cellular transport systems1, protein synthesis1, ribosomal and tRNA synthesis2and eventually the induction of DNA synthesis followed by cell division3,4. Two of the earliest changes observed after growth induction by serum are a transient increase in intracellular cyclic GMP5(10-fold) and a decrease in cyclic AMP (two-to-threefold)5,6,7. It has been suggested that cyclic GMP acts as a positive intracellular signal for cell growth since intracellular cyclic GMP concentrations showed an early transient increase upon growth induction by phytohaemagglutinin whereas no changes were observed in cyclic AMP concentrations8, and additions of high, non-physiological (10−6to 10−4M) concentrations of cyclic GMP can induce substantial increases in DNA synthesis in resting fibroblasts5. Recently a new polypeptide hormone, fibroblast growth factor (FGF), was isolated from bovine pituitary glands9. FGF in combination with the glucocorticoid, hydrocortisone, and a nonspecific carrier protein, bovine serum albumin (BSA), can completely replace exogenously added serum in bringing about all the steps leading to the initiation of DNA synthesis and cell division in some lines of BALB/c 3T3 cells10. Hydrocortisone, as it fails to initiate DNA synthesis alone in the absence of serum11is considered to potentiate the action of FGF9(permissive effect).

The potential calcium-binding protein p9Ka is related to S-100 protein and the vitamin D-dependent intestinal calcium-binding protein. p9Ka accumulates abundantly in cultured rat mammary myoepithelial-like cells but is very much less abundant in the parental cuboidal epithelial cells. p9Ka mRNA is found in normal rat mammary gland, and preliminary experiments suggest that it is found in the mammary myoepithelial cells. A 17-kilobase pair fragment of cloned normal rat DNA contains the gene for p9Ka, but it also contains the gene for two additional polypeptides of molecular mass 6 kDa that are resolved as two isoelectric focusing variants by two-dimensional gel electrophoresis. These two isoelectric focusing variants correspond to two abundant polypeptides present in the cultured myoepithelial cells and probably arise from postsynthetic modification of the product of a single gene. The mRNA for the product of this gene and the p9Ka mRNA are both found in the normal rat mammary gland, but these two mRNAs are differentially expressed in certain tumor-derived rat cell lines.

Prostaglandin (PG) F2alpha added to quiescent Swiss mouse 3T3 cell cultures initiates DNA synthesis and cell proliferation in a small proportion of the cells. Insulin, which markedly potentiates the effect of PGF2alpha on the initiation of cell division when added with PGF2alpha, causes a simultaneous reduction in intracellular cyclic AMP and an increase in cyclic GMP concentrations similar to those observed after serum addition. Prostaglandins E1 and E2 are less effective than PGF2alpha in initiating DNA synthesis and this effect is only observed at higher concentrations. Therefore, some prostaglandins can act as extracellular factors to regulate cell proliferation.

The topographical distribution of type IV collagen, laminin, fibronectin, and the thymocyte differentiation antigen Thy-1 in the basement membrane of the lactating rat mammary gland was investigated. Small cubes of tissue, which had not been subjected to prior fixation or freezing, were incubated with monospecific or monoclonal antibodies to these proteins, and the antibodies were located by an indirect immunoperoxidase staining technique and observed in the electron microscope. The lamina densa stained uniformly with antibodies to type IV collagen and laminin. In addition, both laminin and type IV collagen were present in semiperiodic clusters that traversed the lamina lucida from the cell surface to the lamina densa. Fibronectin was present only in the semiperiodic clusters and not elsewhere in the basement membrane. These clusters were irregularly spaced along the cell surface and heterogeneous in size. It remains to be determined if these three proteins are present in the same clusters. Thy-1 was largely present on the lamina densa and not on the lamina lucida. The Thy-1 staining of the lamina densa occurred in discrete maxima and minima. These maxima occurred in regions adjacent to Thy-1-bearing stromal cells. Thus, the topographical distribution of proteins within a basement membrane varies in a nonrandom manner, and local factors can modify this distribution. We suggest that this topographical variability may play a role in cell recognition and signalling processes that occur across the basement membrane.

We performed immunocytochemical staining of benign, in situ, and malignant breast disease to identify antigens related to the presence of the major parenchymal cell types of the normal breast. Markers for the epithelial cells, antiserum to epithelial membrane antigen, and three monoclonal antibodies (MAb) to milk-fat globule membranes stained most of the inner cells in benign breast lesions, carcinoma in situ, and invasive carcinomas, but the peripheral cells in benign lesions, as well as in carcinoma in situ, were unstained. MAb to epithelium-specific keratin 18 stained the majority of inner cells in benign breast lesions but comparatively fewer such cells in carcinoma in situ and invasive carcinoma. Markers for the myoepithelial cells, antisera, and MAb to smooth muscle actin and vimentin stained most of the peripheral cells in benign breast lesions and in carcinoma in situ but failed to stain virtually any neoplastic cells in invasive carcinomas. Markers for the basement membrane adjacent to the myoepithelial cells, antiserum, and MAb to laminin and Type IV collagen delineated an intact basement membrane around benign lesions and carcinoma in situ, and fragmented structures in 5-10% of invasive carcinomas; the remaining carcinomas were largely unstained. Markers for both myoepithelial and epithelial cells, keratin MAb PKK2 and LP34, stained most of the inner cells in benign lesions but usually only relatively few malignant cells in carcinoma in situ and invasive carcinomas. Markers for the secretory alveolar cell, MAb to beta- and kappa-casein, stained a few isolated cells in benign lesions, many more inner cells in two such lesions in pregnant females, and none in invasive carcinomas. In conclusion, the myoepithelial cell and, under suitable hormonal conditions, the secretory alveolar cell, are retained in most benign lesions, but they are largely lost in invasive carcinomas.

We have determined the relationship between the binding sites for acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) in heparan sulfate (HS) prepared from a panel of mammary cell lines and the ability of the HS to activate aFGF and bFGF in DNA synthesis assays. The ka of the HS for aFGF fell into three groups, whereas the kd (0.0015-0.016 s-1) and the Kd (0.4-8.6 microM) formed a continuum. bFGF possessed a high affinity binding site (Kd 22-30 nM) with a fast ka (320,000-550,000 M-1 s-1), termed "fast/high," and a lower affinity site (Kd 47-320 nM) with a slower ka (35,000-150,000 M-1 s-1), termed "slow/low." Most of the species of HS possessed the latter binding site, which was able to activate bFGF in HS-deficient fibroblasts. However, the HS from the culture medium of the mammary fibroblasts and the myoepithelial-like cells possessed both a fast/high and a slow/low binding site and could not activate bFGF, although it could potentiate the growth-stimulatory activity of aFGF. Treatment of the HS possessing two binding sites for bFGF with heparitinase 1 released oligosaccharides that were able to restore the activity of bFGF in HS-deficient fibroblasts.

A syngeneic, immunocompetent rat mammary model has been employed to investigate the molecular basis of the metastatic phenotype. Transfection of the benign rat mammary epithelial cell line Rama 37 with the gene for p9Ka; a small, rat, calcium-binding protein or DNA from metastatic human cell lines derived from a primary breast carcinoma or a pleural effusion yields transfectants with metastatic capabilities. Transfection of DNA from a benign human mammary cell line, a plasmid containing a cDNA for the human basic fibroblast growth factor (bFGF) gene, or the oncogene EJ-ras-1, fails to yield any transfectants expressing the metastatic phenotype.

Three morphologically distinct major cell types were observed in primary cultures obtained from the mammary parenchyma of glands from virgin rats. These cell types consisted of small cuboidal epithelial cells, larger epithelioid cells and elongated cells. We have investigated the distribution of the basement membrane proteins laminin and type IV collagen, and the intermediate filament proteins vimentin and prekeratin, in these three cell types using immunofluorescence techniques. Antisera to the basement membrane proteins stain the large epithelioid cells and the elongated cells, but do not stain the small cuboidal cells. Polyclonal antiserum to keratin stains all the small cuboidal and large epithelioid cells, but only a small subpopulation of the elongated cells. However, a monoclonal antibody to keratin, LP34, stains only the large cuboidal and a proportion of the elongated cells. Vimentin antiserum fails to stain the small cuboidal cells but stains all the large epithelioid and elongated cells. In addition, peanut lectin, which binds only to ductal lining epithelial cells in the virgin rat mammary gland in vivo after their treatment with neuraminidase, binds to the small cuboidal cells after neuraminidase treatment but not to the other cell types. However, Griffonia simplicifolia agglutinin I, which specifically stains myoepithelial cells in vivo, binds to the large epithelioid and elongated cells but not to the small cuboidal cells. These results suggest that the small cuboidal cells are related to mammary ductal epithelial cells whereas the large epithelial and elongated cells have some characteristics of myoepithelial cells.

After addition of prostaglandin F2alpha (PGF2alpha) and insulin to quiescent cultured 3T3 fibroblasts a constant lag phase of 15 hr occurs before an increased rate of cellular exit from G1 is observed. The latter process follows first order kinetics, which can be quantified by a rate constant k. The temporal relationship of the interactions of PGF2 alpha, insulin, and hydrocortisone to produce alterations in the rate of exit from G1 was investigated. PGF2alpha establishes a constant lag phase and produces alterations in the rate constant k. These two effects can be partially separated by adding two concentrations of PGF2alpha at different times. Insulin fails to establish the lag phase but can stimulate the effect of PGF2alpha when added at any time after PGF2alpha. Hydrocortisone reduces the value of k when added between 0 and 3 hr after PGF2alpha. These results show that the lag phase can beseparated into temporal regions during which hormones can interact to produce changes in the rate of cellular exit from G1.

Transformation of primary cultures of human breast cells with simian virus 40 and clonal selection has yielded single-cell-cloned, epithelial cell lines, as well as myoepithelial-related cell lines. When grown on floating collagen gels, the epithelial cell lines give rise to branching rays of cells, thick fingerlike protrusions, saclike structures, and degenerating areas. The myoepithelial-related cell lines give rise only to the branching rays. Epidermal growth factor stimulates the production of the thick protrusions, whereas cholera toxin stimulates the production of the degenerating areas. Immunocytochemical staining of these cultures using reagents directed against the cell surface-extracellular matrix or the cellular cytoskeleton confirms the epithelial and myoepithelial nature of the cells, and demonstrates that the degenerating areas are undergoing squamous metaplasia. The fingerlike protrusions consist of cords of cells composed of inner, epithelial and outer, myoepithelial-related cells sometimes surrounding a central lumen reminiscent of ducts. The saclike structures resemble alveoli. Ultrastructural analysis confirms the identification of the basic cell types and also identifies indeterminate cells possessing features of both epithelial and myoepithelial cells. It is suggested that the epithelial cell lines represent human mammary stem cells that can undergo processes of morphogenesis and differentiation in vitro to form many of the three-dimensional structures found within the breast.