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Biomedical subjects

J D Black

Publications and source records attributed to J D Black.

At least 19 recordsLinked to original sources
PubMed2001

Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells.

The marine toxin bistratene A (BisA) potently induces cytostasis and differentiation in a variety of systems. Evidence that BisA is a selective activator of protein kinase C (PKC) delta implicates PKC delta signaling in the negative growth-regulatory effects of this agent. The current study further investigates the signaling pathways activated by BisA by comparing its effects with those of the PKC agonist phorbol 12-myristate 13-acetate (PMA) in the IEC-18 intestinal crypt cell line. Both BisA and PMA induced cell cycle arrest in these cells, albeit with different kinetics. While BisA produced sustained cell cycle arrest in G(0)/G(1) and G(2)/M, the effects of PMA were transient and involved mainly a G(0)/G(1) blockade. BisA also produced apoptosis in a proportion of the population, an effect not seen with PMA. Both agents induced membrane translocation/activation of PKC, with BisA translocating only PKC delta and PMA translocating PKC alpha, delta, and epsilon in these cells. Notably, while depletion of PKC alpha, delta, and epsilon abrogated the cell cycle-specific effects of PMA in IEC-18 cells, the absence of these PKC isozymes failed to inhibit BisA-induced G(0)/G(1) and G(2)/M arrest or apoptosis. The cell cycle inhibitory and apoptotic effects of BisA, therefore, appear to be PKC-independent in IEC-18 cells. On the other hand, BisA and PMA both promoted PKC-dependent activation of Erk 1 and 2 in this system. Thus, intestinal epithelial cells respond to BisA through activation of at least two signaling pathways: a PKC delta-dependent pathway, which leads to activation of mitogen-activated protein kinase and possibly cytostasis in the appropriate context, and a PKC-independent pathway, which induces both cell cycle arrest in G(0)/G(1) and G(2)/M and apoptosis through as yet unknown mechanisms.

Acetamides↗
PubMed2001

Sp1 and krüppel-like factor family of transcription factors in cell growth regulation and cancer.

The Sp/KLF family contains at least twenty identified members which include Sp1-4 and numerous krüppel-like factors. Members of the family bind with varying affinities to sequences designated as 'Sp1 sites' (e.g., GC-boxes, CACCC-boxes, and basic transcription elements). Family members have different transcriptional properties and can modulate each other's activity by a variety of mechanisms. Since cells can express multiple family members, Sp/KLF factors are likely to make up a transcriptional network through which gene expression can be fine-tuned. 'Sp1 site'-dependent transcription can be growth-regulated, and the activity, expression, and/or post-translational modification of multiple family members is altered with cell growth. Furthermore, Sp/KLF factors are involved in many growth-related signal transduction pathways and their overexpression can have positive or negative effects on proliferation. In addition to growth control, Sp/KLF factors have been implicated in apoptosis and angiogenesis; thus, the family is involved in several aspects of tumorigenesis. Consistent with a role in cancer, Sp/KLF factors interact with oncogenes and tumor suppressors, they can be oncogenic themselves, and altered expression of family members has been detected in tumors. Effects of changes in Sp/KLF factors are context-dependent and can appear contradictory. Since these factors act within a network, this diversity of effects may arise from differences in the expression profile of family members in various cells. Thus, it is likely that the properties of the overall network of Sp/KLF factors play a determining role in regulation of cell growth and tumor progression.

Animals↗
PubMed2001

Passive stretching does not protect against acute contraction-induced injury in mouse EDL muscle.

A popular part of many athletes pre-game regime is to stretch. We examined whether a pre-injury stretching protocol could prevent acute contraction-induced injury. The in situ extensor digitorum longus (EDL) muscle of an anesthetized mouse (80 mg/kg intra-peritoneal) was used. Damage to the muscle from eccentric contraction-induced injury was quantified by the deficit in tetanic force production, and was not confounded by metabolic fatigue. The force deficits resulting from eccentric contractions alone (E) were compared with the force deficits resulting from a protocol that consisted of a stretch before the eccentric contractions (S + E). The pre-injury stretch was performed to 5% L0 strain, at a velocity of 0.5 mm/s. The muscle was held in the stretch position for 1 min, then slowly released. Eccentric contraction protocols (excursion > or = 24% L0) resulted in pronounced force deficits that increased with the excursion amplitude of the eccentric contraction. The eccentric contractions also resulted in an average right shift of 2 +/- 0.53% in the length-force relationship (t-test, P = 0.0001). The regression lines for the E (eccentric contraction only) and S + E (stretch and eccentric contractions) treatments did not differ from one another for either force deficit (ANCOVA, P = 0.82) or work deficit (ANCOVA, P = 0.12). Therefore, the pre-injury stretch protocol did not reduce the force deficit or the work deficit resulting from contraction-induced injury.

Animals↗
PubMed2001

Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4).

Nonsense-mediated mRNA decay (NMD), also called mRNA surveillance, is an important pathway used by all organisms that have been tested to degrade mRNAs that prematurely terminate translation and, as a consequence, eliminate the production of aberrant proteins that could be potentially harmful. In mammalian cells, NMD appears to involve splicing-dependent alterations to mRNA as well as ribosome-associated components of the translational apparatus. To date, human (h) Upf1 protein (p) (hUpf1p), a group 1 RNA helicase named after its Saccharomyces cerevisiae orthologue that functions in both translation termination and NMD, has been the only factor shown to be required for NMD in mammalian cells. Here, we describe human orthologues to S. cerevisiae Upf2p and S. cerevisiae Upf3p (Caenorhabditis elegans SMG-4) based on limited amino acid similarities. The existence of these orthologues provides evidence for a higher degree of evolutionary conservation of NMD than previously appreciated. Interestingly, human orthologues to S. cerevisiae Upf3p (C. elegans SMG-4) derive from two genes, one of which is X-linked and both of which generate multiple isoforms due to alternative pre-mRNA splicing. We demonstrate using immunoprecipitations of epitope-tagged proteins transiently produced in HeLa cells that hUpf2p interacts with hUpf1p, hUpf3p-X, and hUpf3p, and we define the domains required for the interactions. Furthermore, we find by using indirect immunofluorescence that hUpf1p is detected only in the cytoplasm, hUpf2p is detected primarily in the cytoplasm, and hUpf3p-X localizes primarily to nuclei. The finding that hUpf3p-X is a shuttling protein provides additional indication that NMD has both nuclear and cytoplasmic components.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium.

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

Animals↗
PubMed2000

Protein kinase C-mediated regulation of the cell cycle.

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth/cell cycle progression and differentiation. Increasing evidence from studies using in vitro and in vivo systems points to PKC as a key regulator of critical cell cycle transitions, including cell cycle entry and exit and the G1 and G2 checkpoints. PKC-mediated control of these transitions can be negative or positive, depending on the timing of PKC activation during the cell cycle and on the specific PKC isozymes involved. Most of the mechanistic information available relates to the involvement of this enzyme family in negative regulation of these transitions. Accumulating data indicate that a major target for PKC-mediated inhibition of cell cycle progression is the Cip/Kip cyclin-dependent kinase (cdk) inhibitor p21waf1/cip1. Increased expression of p21waf1/cip1 blocks cdk2 activity in G1 phase, leading to hypophosphorylation of the retinoblastoma protein and inhibition of cell cycle progression into S phase. In G2, p21waf1/cip1 expression blocks cdc2/cyclin B activity, likely through an indirect mechanism involving inhibition of the cdk2/cyclin A complex, and prevents progression into M phase. PKC signaling can also activate a coordinated program of pocket protein regulation leading to cell cycle withdrawal into G0. The molecular events underlying positive regulation of cell cycle progression by PKC signaling remain poorly understood, although there is evidence for a role of the enzyme in promoting G2(r)M progression by phosphorylating lamin B at sites involved in nuclear lamina disassembly. Understanding of the mechanisms underlying PKC-mediated control of the cell cycle is beginning to provide important insight into its role in uncontrolled cell growth and transformation.

Animals↗
PubMed1999

Dynamic association of L-selectin with the lymphocyte cytoskeletal matrix.

L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial venules (HEV). This study examines the biochemical basis and regulation of interactions between L-selectin, an integral transmembrane protein, and the lymphocyte cytoskeleton. Using a detergent-based extraction procedure, constitutive associations between L-selectin and the insoluble cytoskeletal matrix could not be detected. However, engagement of the L-selectin lectin domain by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-derived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytoskeleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosine kinase activity (staurosporine, calphostin C, or genistein), although L-selectin-mediated adhesion of human PBL was markedly suppressed by these agents. Exposure of human PBL or murine pre-B transfectants expressing full-length human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enhanced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimulation and did not support adhesion to HEV. These studies, when taken together with the previously demonstrated interaction between the L-selectin cytoplasmic domain and the cytoskeletal linker protein alpha-actinin, strongly implicate the actin-based cytoskeleton in dynamically controlling L-selectin adhesion.

Antibodies, Monoclonal↗
PubMed1999

Polyamine analogue induction of the p53-p21WAF1/CIP1-Rb pathway and G1 arrest in human melanoma cells.

Although polyamines are well recognized for their critical involvement in cell growth, the cell cycle specificity of this requirement has not yet been characterized with respect to the newly delineated regulatory pathways. We recently reported that polyamine analogues having close structural and functional similarities to the natural polyamines produce a distinct G1 and G2-M cell cycle arrest in MALME-3M human melanoma cells. To determine a molecular basis for this observation, we examined the effects of N1,N11-diethylnorspermine on cell cycle regulatory proteins associated with G1 arrest. The analogue is known to deplete polyamine pools by suppressing biosynthetic enzymes and potently inducing the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Treatment of MALME-3M cells with 10 microM N1,N11-diethylnorspermine caused an increase in hypophosphorylated Rb, which correlated temporally with the onset of G1 arrest at 16-24 h. Rb hypophosphorylation was preceded by an increase in wild-type p53 (approximately 100-fold at maximum) and a concomitant increase in the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (p21; approximately 5-fold at maximum). Another cyclin-dependent kinase inhibitor, p27KIP1, and cyclin D increased slightly, whereas proliferating cell nuclear antigen and p130 remained unchanged. Induction of p21 protein was accompanied by an increase in p21 mRNA, whereas induction of p53 protein was not, suggesting transcriptional activation of the former and posttranscriptional regulation of the latter. SK-MEL-28 human melanoma cells, which contain a mutated p53, failed to induce p53 or p21 and did not arrest in G1. Rather, these cells rapidly underwent programmed cell death within 48 h. Overall, these findings provide the first indication of the cell cycle regulatory pathways by which polyamine antagonists such as analogues might inhibit growth in cells containing wild-type p53 and further suggest a mechanistic basis for differential cellular responses to these agents.

Antineoplastic Agents↗
PubMed1999

Polarized expression of immunoglobulin, spectrin, and protein kinase C beta II occurs in B cells from normal BALB/c, autoimmune lpr, and anti-ssDNA transgenic, tolerant mice.

The rapid redistribution of B cell surface immunoglobulin to a cap upon cross-linking treatment is a well-described phenomenon, the physiological significance of which is unknown. We describe the observation that splenic B cells from unimmunized normal, autoimmune, and tolerant mice express naturally occurring capped immunoglobulin in the absence of exogenous stimulation. The percentage of capped B cells increases to 20% of B cells by age 16 weeks in the progressive autoimmune lpr mouse. Transgenic, tolerant mice expressing lpr-derived genes for ssDNA-binding antibody also demonstrate a large percentage (35-75%) of immunoglobulin-capped splenic B cells. In these capped B cells, protein kinase C beta II, the cytoskeletal proteins spectrin and ankyrin, and the lipophilic probe diI are enriched beneath the site of the immunoglobulin cap. These data suggest that polarization of surface receptors, signaling molecules, anionic phospholipid domains, and cytoskeletal proteins may be an important part of the B cell immune response in vivo.

Animals↗
PubMed1998

Lysosomal sequestration of polyamine analogues in Chinese hamster ovary cells resistant to the S-adenosylmethionine decarboxylase inhibitor, CGP-48664.

CGP-48664, an inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), is presently undergoing Phase 1 clinical trials as an experimental anticancer agent. We have shown previously (D. L. Kramer et al., J. Biol. Chem., 270: 2124-2132, 1995) that Chinese hamster ovary (CHO) cells that are made resistant to the growth inhibitory effects of the drug overexpress AdoMetDC because of a stable gene amplification. Unexpectedly, these same cells (CHO/644) were found to be insensitive to the growth inhibitory effects of N1,N11-diethylnorspermine (DENSPM)-a polyamine analogue also undergoing Phase 1 clinical trials-despite accumulating approximately 5 times more analogue than parental cells. We now report that treatment of CHO/664 cells with DENSPM results in the formation of numerous large cytoplasmic vacuoles, which on the basis of electron microscopy and cytochemical staining seem to be lysosomal in origin. A series of newly established CHO cell lines made differentially resistant to 1, 3, 10, 30, and 100 microM CGP-48664 by chronic exposure were used to demonstrate that vacuole formation correlated with the accumulation of extremely high levels of DENSPM without increasing growth inhibition. These same cells were used to show that AdoMetDC gene overexpression as indicated by mRNA levels was unrelated to vacuole formation; cells resistant to 100 microM CGP-48664 displayed a 170-fold increase in AdoMetDC mRNA levels and formed vacuoles in response to DENSPM, whereas those resistant to 10 microM CGP-48664 displayed a 120-fold increase in AdoMetDC mRNA levels and failed to form vacuoles. Despite accumulating to high intracellular levels, DENSPM was much less effective than spermine at down-regulating ornithine decarboxylase and polyamine transport activities in highly resistant cells. Similarly, DENSPM was less able to induce spermidine/spermine N1-acetyltransferase activity in cells that formed vacuoles than in those that did not. Overall, natural polyamines failed to induce vacuoles and various analogues of DENSPM were used to probe the structural specificity of the effect. The data are consistent with the probability that DENSPM is sequestered to high concentrations in lysosomal vacuoles of CGP-48664-resistant cells and is, therefore, not available to interact with polyamine regulatory sites or to cytotoxically affect cell growth. In addition to implicating the lysosome as a potential new site of CGP-48664 drug action that could be involved in antitumor activity and/or host toxicities, the findings also suggest a potential mechanism of cell resistance to analogues such as DENSPM.

Adenosylmethionine Decarboxylase↗
PubMed1998

Interleukin 15 offers selective protection from irinotecan-induced intestinal toxicity in a preclinical animal model.

Irinotecan (CPT-11) is a chemotherapeutic agent that is active in the treatment of a variety of solid tumor malignancies. Diarrhea represents the most common dose-limiting toxicity that is independent of the schedule of administration. A rat model with dose-limiting toxicity profiles that are similar to those observed in patients treated with CPT-11 was developed and used to evaluate the role of interleukin 15 (IL-15) in the modulation of the therapeutic selectivity of CPT-11 in normal rats and rats bearing advanced colorectal cancer. The maximum tolerated dose and lethal dose (LD) of CPT-11 by i.v. push daily x 3 were 150 and 200 mg/kg/day, respectively. CPT-11 at the LD induced a 93-100% incidence of severe diarrhea and an 86-100% incidence of lethality in treated animals. IL-15, a cytokine with multiple mechanisms of action, was used at a 100 or 400 microg/kg/dose with different schedules of administration (3, 8, and 11 doses, i.p.) to protect against CPT-11-induced toxicity. IL-15 offered complete and sustained selective protection against CPT-11-induced delayed diarrhea and lethality. IL-15 also moderately potentiated the antitumor activity of CPT-11 in rats bearing advanced colorectal cancer. Morphological examination of rat intestinal tissues after treatment with LD of CPT-11 revealed dramatic protection of duodenal and colonic tissue architecture by IL-15. CPT-11 alone produced serious damage to duodenal villi and colonic crypts. The results clearly demonstrated the ability of IL-15 to provide significant protection from CPT-11-induced intestinal toxicity with maintenance of antitumor activity, resulting in an increase in the therapeutic index of CPT-11. The clinical relevance of the results obtained in this model system needs to be confirmed.

Animals↗
PubMed1998

Colonocyte differentiation is associated with increased expression and altered distribution of protein kinase C isozymes.

BACKGROUND & AIMS: Colon cancer cells express reduced levels of protein kinase C (PKC). This study examines the regulation of PKC isozymes in normal colonic epithelium, as a basis for understanding the significance of alterations in this enzyme system in colon carcinogenesis. METHODS: The expression and localization of PKC isozymes in mouse and rat colonocytes at different developmental stages were determined using a combined morphological and biochemical approach. PKC alpha expression was compared in colonic adenocarcinomas and adjacent normal mucosa by immunoblot analysis. RESULTS: Mouse and rat colonocytes express PKC alpha, beta II, delta, epsilon, and zeta. Relatively low levels of these isozymes were detected in proliferating cells of the crypt base, predominantly in the cytosolic compartment. Coincident with colonocyte growth arrest/differentiation, PKC isozyme expression markedly increased in both the cytosolic and, more significantly, in the membrane/cytoskeletal fraction. Colonic tumors express reduced levels of PKC alpha, an isozyme that has been implicated in negative control of intestinal cell growth. CONCLUSIONS: These findings are supportive of a role for certain PKC isozyme(s) in signaling pathways mediating postmitotic events in colonocytes in situ, and suggest that diminished activity of these pathway(s) may contribute to the alterations in growth control/differentiation associated with colonic neoplasia.

Animals↗
PubMed1997

Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21(waf1/cip1) and p27(kip1) and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells.

The molecular mechanisms underlying protein kinase C (PKC) isozyme-mediated control of cell growth and cell cycle progression are poorly understood. Our previous analysis of PKC isozyme regulation in the intestinal epithelium in situ revealed that multiple members of the PKC family undergo changes in expression and subcellular distribution precisely as the cells cease proliferating in the mid-crypt region, suggesting that activation of one or more of these molecules is involved in negative regulation of cell growth in this system (Saxon, M. L., Zhao, X., and Black, J. D. (1994) J. Cell Biol. 126, 747-763). In the present study, the role of PKC isozyme(s) in control of intestinal epithelial cell growth and cell cycle progression was examined directly using the IEC-18 immature crypt cell line as a model system. Treatment of IEC-18 cells with PKC agonists resulted in translocation of PKC alpha, delta, and epsilon from the soluble to the particulate subcellular fraction, cell cycle arrest in G1 phase, and delayed transit through S and/or G2/M phases. PKC-mediated cell cycle arrest in G1 was accompanied by accumulation of the hypophosphorylated, growth-suppressive form of the retinoblastoma protein and induction of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). Reversal of these cell cycle regulatory effects was coincident with activator-induced down-regulation of PKC alpha, delta, and epsilon. Differential down-regulation of individual PKC isozymes revealed that PKC alpha in particular is sufficient to mediate cell cycle arrest by PKC agonists in this system. Taken together, the data implicate PKC alpha in negative regulation of intestinal epithelial cell growth both in vitro and in situ via pathways which involve modulation of Cip/Kip family cyclin-dependent kinase inhibitors and the retinoblastoma growth suppressor protein.

Animals↗
PubMed1996

The relationship between smoking exposure and p53 overexpression in colorectal cancer.

Although epidemiological studies of the relationship between cigarette smoking and colorectal cancer risk have been equivocal, a positive association is consistently found for colorectal adenoma development. We performed an epidemiological study to determine whether p53 protein overexpression, in tumours obtained at the time of resection, is associated with cigarette exposure in colorectal cancer. A total of 163 colorectal cancer cases and 326 healthy controls responded to a standardised questionnaire on colorectal cancer risk factors including detailed information on their history of cigarette smoking. All patients' tumours were analysed immunohistochemically for p53 overexpression using an avidin-biotin immunoperoxidase procedure and polyclonal anti-p53 antibody CM1. Comparison of colorectal cases with controls revealed an elevated risk for ex-smokers (OR = 1.34, 95% CI 0.85-2.12) and current smokers (OR = 1.13, 95% CI 0.63-2.02) when compared with non-smokers. No dose-response relationship was found for total pack-years of smoking (trend test: P = 0.19). However, a trend for total pack-years of smoking was found when p53-positive cases were compared with p53-negative cases suggesting aetiological, heterogeneity (trend test: P = 0.06). Estimating the individual relative risk of developing a p53-positive tumour relative to controls showed no associations for smoking status or total pack-years of smoking. However, when p53-negative cases were compared with controls, an elevated risk was found for ex-smokers (OR = 1.84, 95% CI 1.00-3.37) and current years of smoking (trend test: P = 0.03). Colorectal tumours developing through p53-positive dependent pathways were not associated with smoking exposure. A significant increase in risk was observed for the p53-negative independent pathway with smoking. p53 overexpression appears to be associated with smoking exposure in colorectal cancer.

Adult↗
PubMed1996

Familial and nutritional risk factors for p53 overexpression in colorectal cancer.

Although familial and dietary factors are recognized as important risk determinants for colorectal tumorigenesis, the specific causes of colorectal cancer remain unclear. Studies of p53 genetic alterations have provided clues concerning the etiology of many cancers. This study was designed to determine whether overexpression of the p53 protein is associated with familial and dietary risk factors. Epidemiological data were obtained from 163 colorectal cancer cases and 326 healthy controls. Tumors of all patients were analyzed immunohistochemically for p53 overexpression using an avidin-biotin immunoperoxidase procedure and polyclonal anti-p53 antibody CM1. Of patient tumors, 44.8% showed p53 nuclear reactivity. Colorectal cases versus controls were three times more likely to report a family history of colorectal cancer [odds ratio (OR), 3.12; 95% confidence interval (CI), 1.77-5.52]. Only cruciferous vegetables exhibited a significant inverse association (OR, 0.59; 95% CI, 0.34-1.02; trend test, P = 0.03) for the highest versus lowest quartiles. Both meat and beef displayed an elevated increase in risk. When cases with p53 overexpression (p53 positive) were compared with cases without p53 overexpression (p53 negative), etiological heterogeneity was suggested for family history of colorectal cancer (OR, 0.39; 95% CI, 0.16-0.93), cruciferous vegetables (trend test, P = 0.12), and beef consumption (trend test, P = 0.08). To estimate the individual relative risks for p53-dependent and p53-independent pathways, each p53 subtype was compared with controls. Cruciferous vegetables exhibited a significant association (OR, 0.37; 95% CI, 0.17-0.82; trend test, P = 0.03) when p53 positive cases were compared with controls. When p53 negative cases were compared with controls, a significant increase in risk was observed for family history of cancer (OR, 4.46; 95% CI, 2.36-8.43) and beef (OR, 3.17; 95% CI, 1.83-11.28; trend test, P = 0.006). The p53 (positive) dependent pathway was characterized by an inverse association with cruciferous vegetables, and p53-independent tumors were characterized by family history and beef consumption. These data may indicate the significance of linking epidemiology and molecular biology in assessing specific etiological pathways.

Adenocarcinoma↗
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↗