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M M Ip

Publications and source records attributed to M M Ip.

At least 37 records · Page 2Linked to original sources
PubMed1999

Multiple differentiation pathways of rat mammary stromal cells in vitro: acquisition of a fibroblast, adipocyte or endothelial phenotype is dependent on hormonal and extracellular matrix stimulation.

It has previously been shown that mammary stromal cells possess the ability to maintain a fibroblast-like phenotype or differentiate in vitro into mature adipocytes in a hormone-dependent manner. This paper reports that rat mammary stromal cells can also differentiate into capillary-like structures in vitro when cultured on a reconstituted basement membrane (RBM). The differentiation potential of mammary stromal cells was compared with that of human umbilical vein endothelial cells (HUVEC) and 3T3-L1 preadipocytes. When cultured on plastic, mammary stromal cells, 3T3-L1 and HUVEC maintained a fibroblast-like phenotype. Mammary stromal cells and 3T3-L1, but not HUVEC, differentiated into mature adipocytes when cultured in adipogenic medium. When plated on reconstituted basement membrane, all three cell types began to migrate and organize themselves into an interconnected capillary network. By 18-20 h, mammary stromal cells organized into complex, highly branched capillary-like tubules whereas 3T3-L1 cells and HUVEC formed more simple structures. Cross-sectional analysis demonstrated the presence of an internal lumen. Mammary stromal cells were unique in their ability to progressively develop into a three-dimensional, highly branched network invading the RBM surface. The network formation was enhanced by the presence of vascular endothelial growth factor (VEGF) and was inhibited by the anti-angiogenic drug suramin. Western blotting analysis demonstrated the presence of the endothelial-specific marker flk-1, as well as the presence of the tight-junction-associated protein ZO-1. Mammary stromal cell differentiation into capillary structures was not a terminal state, since these cells were still able to differentiate into adipocytes when exposed to adipogenic medium. These findings suggest that mammary stromal cells differentiate into fibroblasts, adipocytes or vascular structures in a hormone- and substatum-dependent manner, and may explain the dramatic changes in stromal composition during both normal mammary gland development and tumorigenesis.

3T3 Cells↗
PubMed1998

Protein kinase C eta upregulation and secretion during postnatal rat mammary gland differentiation.

The mammary gland has the ability to undergo repeated cycles of tightly regulated postnatal proliferation, differentiation, and apoptosis-mediated regression, providing a model to investigate potential regulators of mammary epithelial growth and differentiation. Protein kinase C eta is a candidate regulator of mammary epithelial differentiation, as increased expression of PKC eta is often observed during the terminal differentiation of many epithelial tissues. In this study, PKC eta expression and localization were characterized during puberty, pregnancy, lactation and involution in isolated rat mammary epithelial cells (MEC), as well as in paraffin-embedded and frozen rat mammary gland sections. By Western blot analysis of whole cell lysates from purified MEC, PKC eta protein expression increased during the shift from resting to a pregnant state. This increased PKC eta protein expression during pregnancy was associated with alveolar rather than ductal development, as immunohistochemical staining for PKC eta was increased in differentiating secretory alveoli, but not ducts. By immunofluorescent staining, PKC eta was stained intensely in an intracellular reticular meshwork throughout the cytosol of alveolar epithelial cells from pregnant mammary gland. During lactation, PKC eta was abundant in apocrine bodies budding from the alveolar epithelium, in the lumen of alveoli, and was present in milk, in association with casein, while being decreased in the cytoplasm of the luminal alveolar epithelium. Staining intensity of alveoli for PKC eta decreased further during involution. Western blotting of subcellular fractions from isolated mammary epithelial cells demonstrated that PKC eta remained associated with the membrane and particulate fractions throughout development. The upregulation of PKC eta in alveolar but not ductal epithelium during pregnancy suggests an association with functional secretory differentiation.

Animals↗
PubMed1997

The epidermal growth factor receptor is not required for tumor necrosis factor-alpha action in normal mammary epithelial cells.

Our laboratory has shown that tumor necrosis factor-alpha (TNF alpha) can regulate normal mammary epithelial cell (MEC) growth, morphogenesis, and, under certain circumstances, functional differentiation in a manner similar to epidermal growth factor (EGF). As TNF alpha has been shown to up-regulate EGF receptor (EGFR) expression and function in other systems, the present studies were undertaken to determine whether TNF alpha action in MEC was indirect through stimulation of the EGFR. An inhibitor of EGFR tyrosine kinase activity, PD158780, failed to block proliferation induced by 40 ng/ml TNF alpha and only partially inhibited growth in response to 2 ng/ml TNF alpha. PD158780 was also unable to suppress the extensive morphological development induced by either TNF alpha concentration. In contrast, the effects of TNF alpha and PD158780 on functional differentiation (i.e. casein accumulation) were time dependent. When measured on day 7 after 48 h of treatment, casein accumulation was unaffected by either concentration of TNF alpha or by PD158780. When assessed on day 21 after 16 days of treatment, however, casein levels were decreased by 40 ng/ml TNF alpha and increased by PD158780. Significantly, this PD158780-induced increase in casein was not observed in MEC that had been treated with both PD158780 and TNF alpha. These results thus suggest that EGFR tyrosine kinase activity is not necessary for TNF alpha action in normal MEC.

Animals↗
PubMed1996

Three-dimensional mammary primary culture model systems.

Model systems have been developed to investigate the complex and coordinated regulation of mammary gland development and transformation. Primary cultures, using newly isolated cells or tissue, are optimal for such studies since, in comparison to immortalized cell lines, the normal signal transduction pathways are presumed to be intact. Three such models are described, including whole organ culture, mammary epithelial cell (MEC) organoids, and MEC-stromal cocultures. Studies using whole-organ culture have the advantage that the normal glandular architecture remains intact, the MEC can undergo lobuloalveolar development and express milk proteins in a hormone dependent manner, and, following hormonal withdrawal, undergo involution. Moreover, transformation of the MEC is readily accomplished. Culture of isolated MEC organoids within an EHS-derived reconstituted basement membrane permits extensive proliferation, branching end bud and alveolar morphogenesis, and accumulation of milk protein and lipid in a physiologically relevant hormone- and growth factor-dependent manner. This model can thus be utilized to investigate the mechanism by which various modulators exert their direct effects on the epithelium. Finally, in view of compelling evidence for stromal-epithelial interactions during normal mammary gland development, and potentially also during the development of malignancy, models in which MEC can be cocultured with enriched populations of stroma offer considerable potential as a tool to understand the nature and mechanisms of the interactions that occur during the various developmental states, and how such interactions may go awry during carcinogenesis.

Animals↗
PubMed1996

Differential binding of mutant glucocorticoid receptors to the glucocorticoid response element of the tyrosine aminotransferase gene.

Glucocorticoid receptors (GCRs) in sublines of the mouse P1798 lymphosarcoma that are sensitive (S) or resistant (R) to glucocorticoid-induced cell lysis were examined for their ability to bind to a single glucocorticoid responsive element (GRE). Mobility shift assays detected two specific complexes that were identical in both S and R cellular extracts. Antibodies against the GCR N-terminus supershifted complexes, suggesting that the 97 kDa wild-type GCR (WT-GCR) in S cells, and the variant, 97 kDa non-steroid-binding GCR (NSB-GCR) in R cells were components of both complexes. Sephacryl S300 gel filtration column fractions containing the WT-GCR and NSB-GCR formed complexes with the GRE, while fractions containing a second GCR variant in R cells, the 45 kDa steroid-binding truncated GCR (TR-GCR), did not. Southwestern blotting detected a GRE-binding, 97 kDa protein band in both S and R extracts. A 45 kDa band was not detected. UV crosslinking of protein to DNA revealed protein in the range of 92-120 kDa crosslinked to the GRE in both S and R extracts. No crosslinking was detected at 45 kDa. Strong interaction of the NSB-GCR with GREs and lack of binding of the TR-GCR to single GREs illustrate a complex receptor system in the P1798 lymphosarcoma.

Acrylic Resins↗
PubMed1996

Tumor necrosis factor-alpha: a multifunctional regulator of mammary gland development.

To determine whether tumor necrosis factor-alpha (TNF alpha) plays a physiological role in normal mammary gland development, TNF alpha and TNF receptor expression were measured in epithelial cells (MEC) isolated from mammary glands of virgin, pregnant, lactating, and postlactational (day 7 of involution) rats. TNF alpha messenger RNA (mRNA) increased significantly during pregnancy, then decreased during lactation and involution. The 26-kDa transmembrane form of TNF alpha protein, undetectable in MEC from virgin rats, increased throughout pregnancy and lactation, and disappeared during involution. In contrast, p55 TNF receptor (TNFR) mRNA levels peaked in early lactation and declined thereafter, whereas p75 TNFR mRNA levels rose steadily through lactation. Using agonistic antibodies specific to either TNFR, the individual roles of each TNFR were investigated in MEC in primary culture. The p55 TNFR was found to be the sole mediator of TNF alpha-induced proliferation. Intriguingly, the two receptors were found to have opposing effects on functional differentiation (casein accumulation), with inhibition occurring through the p55 receptor and stimulation through the p75 receptor. Taken together, these results suggest that TNF alpha plays a role in the growth and development of the mammary gland. In addition, both TNF receptors are important for TNF alpha function and may mediate different effects.

Animals↗
PubMed1995

Prolactin and epidermal growth factor regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture.

The epithelial cell-specific effects of prolactin and epidermal growth factor (EGF) on the development of normal rat mammary epithelial cells (MEC) were evaluated using a three dimensional primary culture model developed in our laboratory. Non-milk-producing MEC were isolated as spherical end bud-like mammary epithelial organoids (MEO) from pubescent virgin female rats. The cultured MEO developed into elaborate multilobular and lobuloductal alveolar organoids composed of cytologically and functionally differentiated MEC. Prolactin (0.01-10 micrograms/ml) and EGF (1-100 ng/ml) were each required for induction of cell growth, extensive alveolar, as well as multilobular branching morphogenesis, and casein accumulation. MEO cultured without prolactin for 14 days remained sensitive to the mitogenic, morphogenic, and lactogenic effects of prolactin upon subsequent exposure. Similarly, cells cultured in the absence of EGF remained sensitive to the mitogenic and lactogenic effects of EGF, but were less responsive to its morphogenic effects when it was added on day 14 of a 21-day culture period. If exposure to prolactin was terminated after the first week, the magnitude of the mitogenic and lactogenic effects, but not the morphogenic response was decreased. Removal of EGF on day 7 also reduced the mitogenic response, but did not have any effect on the magnitude of the lactogenic or morphogenic responses. These studies demonstrate that physiologically relevant development of normal MEC can be induced in culture and that this model system can be used to study the mechanisms by which prolactin and EGF regulate the complex developmental pathways operative in the mammary gland.

Animals↗
PubMed1995

Hydrocortisone and progesterone regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture.

The mechanisms of action of, and resistance to, the steroidal regulators of normal mammary epithelial and breast cancer cell development are only partially understood. A major obstacle to research progress has been the difficulty in supporting physiologically relevant development of normal mammary epithelial cells (MEC) under defined serum-free conditions. A primary culture system was developed in our laboratory that permits nonfunctional rat MEC to undergo extensive proliferation, functional differentiation, as well as multilobular and lobuloductal branching alveolar morphogenesis. In the studies reported here, the contributions of hydrocortisone and progesterone during the coordinate induction of cellular proliferation, organoid morphogenesis, and functional capacity were assessed. Hydrocortisone (0.1-10 microgram/ml) induced alveolar and multilobular branching morphogenesis, suppressed lobuloductal branching morphogenesis, and enhanced casein accumulation. Hydrocortisone also played a role in maintaining alveolar as well as multilobular branching morphogenesis and casein levels. Progesterone (0.01-1 microgram/ml) induced cellular proliferation as well as multilobular and lobuloductal branching morphogenesis, and suppressed casein accumulation. At a supraphysiological concentration (10 micrograms/ml), progesterone inhibited cell growth, alveolar branching morphogenesis, and casein accumulation. MEC cultured without progesterone for up to 1 week retained the ability to respond when subsequently exposed to this steroid. Reversibility studies suggested that progesterone was required for the induction, but not the maintenance of the mitogenic, morphogenic, and lactogenic effects. This physiologically relevant primary culture system can be used to study the factors that regulate steroid responsiveness as well as the cross-talk between steroid and growth factor receptor signaling pathways in normal MEC and breast cancer cells.

Animals↗
PubMed1995

Charge heterogeneity in wildtype and variant glucocorticoid receptors.

Two-dimensional electrophoresis was used to examine charge heterogeneity in glucocorticoid receptors (GCRs) from sublines of the thymic-derived, mouse P1798 lymphosarcoma which were sensitive (S) or resistant (R) to glucocorticoid-mediated apoptosis. Previous work had identified the 97 kDa wildtype GCR (WT-GCR) in S cells and two variant GCRs in R cells: a 45 kDa, steroid-binding truncated GCR (TR-GCR), and a 97 kDa non steroid-binding GCR (NSB-GCR). Using denaturing isoelectric focusing, we now show that S cells as well as adult mouse thymus gland also express the NSB-GCR at pI 5.6 in addition to the WT-GCR which resolves between pH 5.9-7.1. Thus, the NSB-GCR is detected in steroid-sensitive cells and is not unique to R cells. Separation of receptors by native isoelectric focusing suggested that the TR-GCR in R cells resolved at a single, high pI (8.1) relative to the WT-GCR which resolved in a broad range (pI 5.8-8.0). The high pI of the TR-GCR may alter its functional activity thereby contributing to the resistance phenotype.

Amino Acid Sequence↗
PubMed1995

Identification and localization of steroid-binding and nonsteroid-binding forms of the glucocorticoid receptor in the mouse P1798 lymphosarcoma.

Glucocorticoid receptors (GCRs) were characterized in sublines of the mouse P1798 lymphosarcoma that are sensitive (S) or resistant (R) to glucocorticoid-mediated apoptosis. Previous work had identified two steroid-binding GCRs in S and R cells: a 97 kDa wild-type GCR in S cells (WT-GCR), and a 45 kDa truncated GCR in R cells (TR-GCR). A third GCR, a 97 kDa nonsteroid-binding GCR (NSB-GCR), was also identified in R cells. Using subcellular fractionation and Western blotting, we now show that in contrast to the WT-GCR which is localized in both the cytoplasm and nucleus of S cells, the NSB-GCR is localized predominantly in R cell nuclei. Moreover, gel filtration chromatography revealed that treatment with 400 mM NaCl and heat did not significantly alter the Stokes radius of the NSB-GCR suggesting that this receptor is not present in a heterooligomeric complex with other proteins. The TR-GCR was localized predominantly in the soluble cytoplasmic fraction but also in the crude membrane fractions of R cell nuclei, suggesting that this receptor is tightly associated with nuclear structures. It was not detected in the soluble nuclear fraction. Unexpectedly, a 45 kDa nonsteroid-binding immunoreactive protein was detected in crude membrane fractions of S cells. These studies describe a complex GCR system in the P1798 lymphosarcoma that necessitates a further consideration of glucocorticoid signaling in S and R cells.

Amino Acid Sequence↗
PubMed1995

Modulation of normal mammary epithelial cell proliferation, morphogenesis, and functional differentiation by retinoids: a comparison of the retinobenzoic acid derivative RE80 with retinoic acid.

The ability of retinoids to modulate the proliferation as well as the morphological and functional differentiation of normal mammary epithelial cells isolated from pubescent female virgin rats was evaluated in serum-free primary culture. The retinobenzoic acid derivative RE80, present continuously or for only a limited time in culture, inhibited proliferation with an IC50 of less than 10(-10) M. In contrast, all-trans-retinoic acid (RA) inhibited proliferation with an IC50 of approximately 10(-8) M. In addition to effects on proliferation, RE80 and RA stimulated end bud colonies to differentiate to lobular alveolar colonies, inhibited alveolar budding, and suppressed the outgrowth of squamous colonies. Both retinoids also markedly stimulated functional differentiation, as assessed by accumulation of the major milk protein casein, and stimulated the synthesis of a approximately 73- to 74-kilodalton protein identified as a member of the transferrin family. Moreover, both retinoids stimulated cell death in the differentiated cell population. RE80 was approximately 100-fold more potent than RA for all of these effects. These data suggest that several mechanisms may contribute to the chemopreventive and/or therapeutic efficacy of retinoids in breast cancer, including inhibition of proliferation, stimulation of cell death, and/or induction of differentiation.

Animals↗
PubMed1995

Interaction of retinoids with steroid and peptide hormones in modulating morphological and functional differentiation of normal rat mammary epithelial cells.

The interaction of the retinoid RE80 with the lactogenic and mammogenic regulators of mammary gland development was investigated using a mammary epithelial cell (MEC) primary culture model in which cells from young virgin rats were cultured within a reconstituted basement membrane using defined serum-free medium. RE80 (10(-10) M) was able to substitute completely for epidermal growth factor and partially for hydrocortisone in stimulating both morphological and functional (casein accumulation) differentiation of the MEC. In contrast, the requirement of PRL for both differentiation processes was absolute. Furthermore, RE80 was found to abrogate the inhibitory effect of progesterone on casein accumulation and to act as an antiprogestin in terms of morphological effects. Under optimal medium conditions, RE80 also inhibited cell proliferation. This inhibition did not require epidermal growth factor, hydrocortisone, progesterone, or PRL, but, unexpectedly, was enhanced in medium deficient in or lacking hydrocortisone. Additionally, RE80 induced the death of differentiated MEC, an effect that was found to require hydrocortisone. These results suggest that retinoids may modulate transcription of the casein gene family, either directly by activation of the binding of retinoic acid receptors to the casein promoter or indirectly by modulation of the effects of other hormones. The antiproliferative effect of retinoid may also be direct or indirect by virtue of down-regulation of the receptors for one of the mitogenic hormones, possibly progesterone.

Animals↗
PubMed1994

Phorbol 12-myristate 13-acetate stimulates proliferation and ductal morphogenesis and inhibits functional differentiation of normal rat mammary epithelial cells in primary culture.

The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on proliferation and differentiation of normal mammary epithelial cells from 50-day-old virgin rats was investigated using a model system that allows for full morphological and functional development of the cells. In this model, mammary epithelial cells are grown within a reconstituted basement membrane in a defined serum-free medium. PMA at a concentration of 10(-6) M effected translocation of protein kinase C from cytosol to membrane. At the same concentration, it stimulated cell proliferation both in the presence and absence of EGF, and this stimulation was observed even when PMA exposure was limited to 15 min at the time of each media change. In contrast to the effect on proliferation, PMA at concentrations of 10(-7) and 10(-6) M inhibited functional differentiation as assessed by casein accumulation. Phorbol 12,13-dibutyrate at 10(-6) M also stimulated proliferation and inhibited casein accumulation and was more effective than PMA in both cases. In contrast, the nonactive tumor promoter 4-alpha PMA had no effect on either proliferation or differentiation. One of the most striking effects of PMA was its ability to stimulate an atypical ductal morphogenesis, as manifested by the formation of intricate web-like colonies, and to inhibit the development of the well-differentiated alveolar-like multilobular colonies. PMA was also shown to completely suppress the growth of the squamous-like colonies that develop when EGF is absent or deficient. These effects of phorbol esters in mammary epithelial cells to stimulate proliferation, inhibit functional differentiation, and stimulate the development of ductal colonies are consistent with the suggestion that the signal transduction pathways evoked by PMA could act to stimulate the growth of initiated cells or render normal cells more sensitive to carcinogen.

Animals↗
PubMed1993

Casein accumulation by rat mammary epithelial cells grown within a reconstituted basement membrane is modulated by fatty acids in a hormone- and time-dependent manner.

The mammary gland is under complex regulation involving the participation of hormones, growth factors, and stromal components, including lipids. Our laboratory has developed a unique primary culture system that allows undifferentiated mammary epithelial cells from immature virgin rats to proliferate and differentiate to an extent equivalent to the lactating mammary gland. Using this model system we have examined the effects of the unsaturated fatty acids oleate and linoleate on mammary epithelial cell proliferation as well as both morphological and functional differentiation. Neither fatty acid showed any effect on cell proliferation whether added to cells in the presence of optimal serum-free medium or under suboptimal conditions of epidermal growth factor (EGF) and prolactin. Morphological differentiation also was not affected by fatty acid addition under either optimal or suboptimal conditions, although a decrease was observed when medium depleted in EGF and prolactin was compared to optimal medium. The notable finding in this study was that both oleate and linoleate modulated functional differentiation, as assessed by casein accumulation, in a time- and hormone-dependent manner. At early times in culture, casein levels were stimulated by both oleate and linoleate; this effect was most dramatic under suboptimal conditions of prolactin and EGF. In marked contrast, however, linoleate decreased casein levels by approximately 50% in optimal medium, at all concentrations tested, after at least 7 days in culture. This decrease was also observed in suboptimal medium, although the concentration of EGF and prolactin influenced the extent of the reduction. Although the mechanism is currently unknown, it is tempting to speculate that the cellular and biochemical events that result in linoleate-induced inhibition of functional differentiation may also be involved in the tumor-enhancing properties of this fatty acid.

Animals↗
PubMed1993

Effects of specific fatty acids on prolactin-induced NB2 lymphoma cell proliferation.

Nb2 rat lymphoma cells are dependent on prolactin (PRL) for growth. Membrane lipid composition of Nb2 cells undergoes rapid modification when these cells are grown in culture media supplemented with specific fatty acids. Since the actions of PRL are mediated through specific membrane receptors, the following studies were conducted to characterize the lipid-dependent events involved in fatty acid modulation of PRL-induced cell proliferation. Nb2 cells were grown in suspension cultures in control or fatty acid-supplemented media, in the presence of various doses of PRL. PRL-induced cell growth was significantly enhanced by arachidonate, but significantly attenuated by stearate supplementation of the culture media. A direct relationship was observed between the concentration of specific fatty acid added to the culture media and the magnitude with which this fatty acid was incorporated into Nb2 cell membranes, as determined by gas chromatography. Acute treatment with phorbol ester enhanced Nb2 cell growth in control media and reversed the attenuating effects of membrane stearic acid enrichment. However, PRL-induced Nb2 cell growth was similar with or without the presence of phorbol ester, when cells were grown in media supplemented with arachidonate. Addition of protein kinase C (PKC) inhibitors to control and fatty acid-supplemented media resulted in a dose-dependent inhibition of PRL-induced Nb2 cell proliferation. These results suggest that lipid modulation of Nb2 mitogenic-responsiveness to PRL is mediated through alterations in PKC activation.

Animals↗
PubMed1992

Regulation of proliferation of rat mammary tumor cells by inhibitors of cyclooxygenase and lipoxygenase.

The studies described herein were undertaken with the objective of determining the effect of modulation of arachidonic acid metabolism on proliferation of rat mammary tumor cells in culture. The TMT-081 rat mammary tumor cell line was shown to metabolize [14C]arachidonic acid to thromboxane B2, PGE2, PGF2 alpha, and 12- and/or 15-HETE. Furthermore, synthesis of these eicosanoids was physiologically significant since each metabolite stimulated DNA synthesis. Both cyclooxygenase and lipoxygenase inhibitors suppressed cell growth and modulated eicosanoid synthesis in a concentration-dependent manner. Thin layer chromatography showed that the production of cyclooxygenase metabolites (thromboxane B2, PGE2 and PGF2 alpha) by TMT-081 cells was inhibited by indomethacin, ibuprofen, BW755C and timegadine. The lipoxygenase inhibitor NDGA was also shown to inhibit thromboxane B2 synthesis, but increased PGE2 and PGF2 alpha syntheses; esculetin, a lipoxygenase inhibitor in other systems, increased all the cyclooxygenase products. The production of the lipoxygenase products 12- and/or 15-HETE was inhibited by NDGA, increased by indomethacin and ibuprofen, but not affected by esculetin, BW755C and timegadine in this cell line. Changes in eicosanoid profile were observed before drug-induced inhibition of cell growth suggesting that the balance of the various eicosanoids may be a critical determinant of cell proliferation. However, since drug-induced effects on arachidonic acid profile occurred at concentrations lower than that necessary for inhibition of cell growth, the effects of these inhibitors on other biochemical pathways affecting cell growth cannot be ruled out.

Animals↗
PubMed1992

Regulation of rat mammary epithelial cell proliferation and differentiation by tumor necrosis factor-alpha.

The effect of tumor necrosis factor-alpha (TNF alpha) on the growth and differentiation of normal rat mammary epithelial cells was evaluated using a model system in which cells were grown within a reconstituted basement membrane under defined serum-free medium conditions. TNF alpha (5-10,000 U/ml) stimulated mammary epithelial cell proliferation both under conditions previously considered optimal for their growth as well as in medium deficient in epidermal growth factor (EGF). Moreover, TNF alpha could completely substitute for EGF for cell proliferation. Under optimal conditions, TNF alpha had no effect on morphological differentiation, but in medium either lacking or deficient in EGF or when suboptimal reconstituted basement membrane was used, TNF alpha (5-100 U/ml) had a marked stimulatory effect on lobular and ductal morphogenesis. The effect of TNF alpha on functional differentiation, as assessed by casein production, was more complex. In optimal lactogenic medium, TNF alpha (10-10,000 U/ml) inhibited casein production. In the absence of EGF, however, the effect of TNF alpha appeared to follow a bell-shaped curve. Thus, omission of EGF per se resulted in a marked suppression of casein production, possibly secondary to an inhibition of morphological development. At low concentrations (approximately 5 U/ml), TNF alpha stimulated casein production in parallel to its stimulation of morphological differentiation, although not to the same extent as in medium containing optimal levels of EGF. However, once maximal stimulation of morphogenesis had been achieved, further increasing the TNF alpha concentration from 5 to 100 U/ml resulted in a concentration-dependent inhibition of casein production. This suggests that TNF alpha may have a direct inhibitory effect on casein gene expression. In summary, this is the first study to report that the multifunctional cytokine TNF alpha is a potential regulator of the growth and development of the mammary gland.

Animals↗