Biomedical subjects

R Dahiya

Publications and source records attributed to R Dahiya.

At least 91 records · Page 5Linked to original sources

PubMed1996

P53 tumour-suppressor gene mutations are mainly localised on exon 7 in human primary and metastatic prostate cancer.

Mutations in the p53 tumour-suppressor gene are among the most common genetic alterations in human cancers. In the present study we analysed the mutations in the p53 tumor-suppressor gene in 25 primary and 20 metastatic human prostate cancer specimens. DNA extracted from the paraffin-embedded sections was amplified by hot-start polymerase chain reaction, and p53 gene mutations in the conserved mid-region (exons 4-9) were examined using single-strand conformation polymorphism (SSCP) analysis and immunohistochemistry. In the present study, we used a novel hot-start PCR-SSCP technique using DNA Taq polymerase antibody, which eliminates primer-dimers and non-specific products. Because of this new technique, the results of PCR-SSCP showed very high resolution. Polymerase chain reaction products were sequenced directly for point mutations for the p53 gene. Mutations were found in 2 out of 25 primary prostate cancers (8%) and 4 out of 20 metastatic cancers (20%). Mutations were observed exclusively in exon 7 and not in exons 4, 5, 6, 8 or 9. Nuclear accumulation of p53 protein, determined by immunohistochemistry, correlated with the degree of metastasis in prostatic cancer.

Adenocarcinoma↗

PubMed1996

Smooth muscle-epithelial interactions in normal and neoplastic prostatic development.

A hypothesis is proposed that unifies prostatic developmental biology and carcinogenesis. The foundation of this hypothesis is the reciprocal interaction of epithelium and mesenchyme during prostatic development followed thereafter by a reciprocal homeostatic interaction of epithelium and smooth muscle in adulthood. This smooth muscle-epithelial cell interaction is perturbed during prostatic carcinogenesis with adverse sequelae for both epithelium and smooth muscle. The following sequence of events is proposed to occur: (1) Under the influence of androgens, urogenital sinus mesenchyme (UGM) induces urogenital sinus epithelium to undergo prostatic ductal morphogenesis and differentiation. (2) As prostatic epithelium differentiates, it in turn signals the UGM to differentiate into smooth muscle cells that closely surround the epithelial ducts. Differentiation of prostatic smooth muscle requires both an inductive signal from epithelium and androgens. (3) Once formed, prostatic smooth muscle participates in reciprocal homeostatic interactions. We propose that prostatic smooth muscle under the influence of androgens signals to prostatic epithelium to maintain epithelial differentiation and to repress epithelial proliferation, while prostatic epithelium signals to prostatic smooth muscle to maintain smooth muscle differentiation. In adulthood, homeostasis is maintained through reciprocal interactions between smooth muscle and epithelial cells with minimal proliferation of either cell type. Prostatic carcinogenesis, which is initiated following genetic damage to prostatic epithelium, is surmised to involve a sequential disruption in these reciprocal homeostatic interactions with ensuing dedifferentiation of both the emerging prostatic carcinoma cells and smooth muscle. Thus, following genetic insult to prostatic epithelium the epithelium fails to signal appropriately to the smooth muscle, which then begins to dedifferentiate. As smooth muscle differentiation begins to deviate, signaling from prostatic smooth muscle to prostatic epithelium becomes anomalous resulting in progressive loss of control over epithelial differentiation and proliferation. During progression of prostatic carcinogenesis a vicious cycle is established in which both prostatic epithelium and smooth muscle dedifferentiate. This hypothesis is based on the ontogeny of prostatic smooth muscle differentiation during development and by the fact that the amount of smooth muscle progressively diminishes in human prostatic adenocarcinomas during progression from low- to high-grade cancers. Finally, in experimental tissue recombinants in which various normal or neoplastic prostatic epithelia were grown in combination with embryonic rat UGM, only normal (non-neoplastic) epithelia were capable of inducing differentiation of prostatic smooth muscle in UGM. Based on several lines of evidence, it is now apparent that smooth muscle-epithelial interactions are the operative cell-cell interaction in the postnatal prostate which plays a key role in regulating epithelial differentiation, proliferation and carcinogenesis.

Animals↗

PubMed1996

Epithelial development in the rat ventral prostate, anterior prostate and seminal vesicle.

The prostate and seminal vesicle (SV) are androgen-dependent secretory glands of the male genital tract. The epithelial cells of these glands produce the bulk of the seminal secretions. The objective of the present study was to examine the ontogeny of cytokeratin and androgen receptor (AR) expression in the rat SV, anterior prostate (AP) and ventral prostate (VP). The study utilized organ culture to examine the effects of androgens on the development of these markers and castration of adult rats to examine androgenic effects on their maintenance. Tissues were examined from 14 days of gestation to adulthood. The SV was a tubular organ from its inception while the prostate formed from solid epithelial cords. These prostatic buds canalized in a proximal to distal manner starting at day 1 postnatal in the VP and day 5 in the AP. The expression of cytokeratins and AR was visualized by immunocytochemistry. In all three glands keratins 5, 7, 8, 14, 18 and 19 were initially uniformly expressed in all epithelial cells. In the SV, segregation of cytokeratins between the luminal and basal cell types started at 4 days postnatally with keratin 7 localizing to basal cells. Five days after birth, keratins 5 and 14 were also localized to the basal epithelium, while keratins 8 and 18 were only expressed by luminal cells, Keratin 19 was expressed in all epithelial cells throughout development and into adulthood. In the VP and AP the same pattern of cytokeratin segregation occurred as in the SV. Epithelial differentiation occurred in a proximal to distal fashion in the prostate. In the proximal VP ducts keratins 7 and 14 were basally localized by 2 days postnatally, while keratin 5 did not clearly segregate to basal cells until day 9 after birth. In the AP keratin 14 was basally localized by 1 day postnatal but keratin 5 and 7 did not colocalize to the basal cells until days 9 and 12, respectively. AR were expressed in the epithelium of the urogenital sinus from 19 days of gestation. At 19 and 20 days of embryonic development AR-negative prostatic buds were seen emerging from the AR-positive urogenital sinus epithelium. By birth AR were detectable in the epithelium of both prostatic lobes and the SV. The role of androgens in the development of the prostatic and SV epithelium was investigated in a serum-free organ culture system. These experiments showed that differentiation of prostatic and SV luminal and basal epithelial cell types was accelerated as compared to the in vivo situation in the presence of androgens, and did not occur in their absence. Following castration of adult animals the prostate and SV regressed with preferential loss of luminal epithelium. The relative numbers of basal cells was increased, though some flattened cells expressing a luminal cell pattern of cytokeratins were still observed. AR were detected in the prostatic and SV epithelium of long-term castrated animals. In summary, the rat prostate was found to be derived from undifferentiated solid epithelial cords. Canalization occurred concurrent with the differentiation of clear epithelial subtypes. Epithelial AR were expressed from around the time of birth and expression levels increased with age. The SV was canalized from its inception but likewise was derived from an undifferentiated epithelial precursor.

Age Factors↗

PubMed1996

Stromal development in the ventral prostate, anterior prostate and seminal vesicle of the rat.

The prostate and seminal vesicle (SV) are androgen-dependent secretory glands of the male genital tract. They produce the bulk of the seminal secretions. The object of the present study was to examine and document the ontogeny of stromal maturation in the rat anterior and ventral prostate and SV. These organs have a loosely organized cellular mesenchyme during fetal development. During prostatic development the mesenchyme condensed to form smooth muscle sheaths immediately surrounding the epithelium, with looser connective tissue between individual ducts. In the SV, a loose connective tissue layer called the lamina propria lies between the epithelium and developing muscle. Smooth muscle alpha-actin, myosin, desmin, laminin, vinculin, vimentin and androgen receptor (AR) expression were examined by immunocytochemical methods during the pre- and postnatal developmental periods. The first marker to be detected was vimentin, which was initially found throughout the mesenchyme. During development vimentin became mostly restricted to the interductal tissue of the prostate and the lamina propria of the SV. Smooth muscle markers were expressed in an orderly sequence in a proximal to distal manner along prostatic ducts, from the urethra towards the tips. Expression of alpha-actin was followed by vinculin, myosin, desmin, and laminin. These markers became localized to the developing smooth muscle sheaths and were not expressed in the interductal tissue of the prostate or the lamina propria of the SV. Organ culture experiments demonstrated that androgens were required for the differentiation of smooth muscle sheaths. Castration of adult rats demonstrated that androgens were required to maintain smooth muscle differentiation. In castrates, the stroma was relatively thicker but less dense than in intact animals. Following castration, expression of the smooth muscle markers was lost sequentially in the reverse order of their expression during development. In long-term castrates alpha-actin, vimentin and a small amount of vinculin were detected. AR were first detected in the urogenital sinus mesenchyme immediately surrounding the epithelium at 16 days of gestation. As development progressed expression of AR became more widespread, and postnatally was found throughout the mesenchyme. As maturation of smooth muscle occurred, stromal expression of AR became localized to the muscular sheath immediately surrounding the epithelium. In the prostate the interductal connective tissue displayed very low levels of AR expression. In the SV, AR were also observed in the lamina propria. In summary, stromal differentiation and dedifferentiation in the rat prostate and SV were found to be androgen-dependent processes with ordered sequential ontogenic expression of specific markers.

Actins↗

PubMed1996

Inhibitory effects of retinoic acids on androgen-dependent development of neonatal mouse seminal vesicles in vitro.

The effects of retinoic acids (RAs) on development of seminal vesicles (SVs) of neonatal mice were investigated in vitro. SVs from 0-day-old male mice were cultured for 2-6 days in serum-free, chemically defined medium containing transferrin and BSA supplemented with 5alpha-dihydrotestosterone (DHT; 10(-8) M) and insulin (10 microg/ml), alone and in combination. Before culture, SVs from 0-day-old mice consisted of an unbranched epithelium surrounded by mesenchyme. SVs cultured in medium with DHT plus insulin or DHT alone formed numerous epithelial branches after day 2 of culture, whereas epithelial branching did not occur in SVs cultured with insulin alone. All-trans-RA or 13-cis-RA (10(-9)-10(-6) M) added to medium containing DHT plus insulin or DHT alone inhibited epithelial branching in a dose-dependent manner. This inhibitory effect was reversible after removal of the retinoids from the medium on day 4 of culture. These RAs also decreased [3H]thymidine labeling indexes of both epithelium and mesenchyme of SVs cultured in medium with DHT plus insulin or DHT alone and inhibited the increase in their protein contents. 9-Cis-RA was less inhibitory than all-trans-RA or 13-cis-RA on epithelial branching, [3H]thymidine labeling indexes of epithelium and mesenchyme, and protein content of SVs cultured in medium with DHT and insulin. In the absence of DHT (insulin alone), all-trans-RA did not affect either the [3H]thymidine labeling indexes of epithelium and mesenchyme or the protein content of cultured SVs. Reverse transcriptase-PCR demonstrated strong expression of transcripts for mouse RA receptors (RARalpha, RARgamma, and RXRalpha), with lower levels of expression of RARbeta, RXRbeta, and RXRgamma in neonatal SVs. The present results indicate that RAs reversibly inhibit androgen-dependent development of neonatal mouse SVs, most likely through RARs.

Animals↗

PubMed1995

Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell line: BPH-1.

This report describes the development and characterization of an epithelial cell line (BPH-1) from human prostate tissue obtained by transurethral resection. Primary epithelial cell cultures were immortalized with SV40 large T antigen. One of the isolated clones was designated BPH-1. These cells have a cobblestone appearance in monolayer culture and are non-tumorigenic in nude mice following subcutaneous injection or subrenal capsule grafting. They express the SV40 large T antigen and exhibit increased levels of p53, as determined by immunocytochemistry. Cytogenetic analysis by G-banding demonstrated an aneuploid karyotype with a modal chromosome number of 76 (range 71 to 79, n = 28) and 6 to 8 marker chromosomes. Some structurally rearranged chromosomes were observed, but the Y chromosome was normal. The expressed cytokeratin profile was consistent with a prostatic luminal epithelial cell. This profile was the same as that of primary prostatic epithelial cultures from which the BPH-1 cells were derived. In serum-free culture in plastic dishes epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, fibroblast growth factor (FGF) 1 (aFGF), and FGF 7 (KGF) induced increased proliferation in these cells whereas FGF 2 (bFGF), TGF-beta 1, and TGF-beta 2 inhibited proliferative activity. Testosterone had no direct effect on the proliferative rate of BPH-1 cells. 5 alpha-Reductase, 3 alpha-hydroxysteroid oxidoreductase, and 17 beta-hydroxy-steroid oxidoreductase activities were detected in BPH-1 cells. Expression of androgen receptors and the secretory markers, prostate specific antigen and prostatic acid phosphatase, were not detectable by immunocytochemistry, biochemical assay, or RT-PCR analysis.

Aged↗

PubMed1995

Inhibition of mucin synthesis by benzyl-alpha-GalNAc in KATO III gastric cancer and Caco-2 colon cancer cells.

Previous studies from our laboratory have shown that benzyl-alpha-GalNAc inhibits the glycosylation of mucin in colon cancer cells. In this study, we determined whether benzyl-alpha-GalNAc inhibits mucin glycosylation in KATO III gastric cancer cells. We also examined its effects on expression of mucin antigens, and compared the mucins made by KATO III with those of a colonic cancer cell line, Caco-2. Results of these experiments suggest that benzyl-alpha-GalNAc (2 mM) inhibited [3H]glucosamine labelling of mucins by 82% in KATO III and by 70% in Caco-2. For both cell lines, the mucin secreted in the presence of benzyl-alpha-GalNAc was less acidic. Both cell lines secreted benzyl-oligosaccharides, but those from KATO III (8-9 sugars) were larger than those from Caco-2 (6-7 sugars). In mucins purified from the medium of treated cells, peripheral carbohydrate antigens (sialyl Lex in KATO III and terminal fucose in Caco-2) were decreased (compared with control), while core carbohydrate antigens (T antigen in both cell lines and sialyl Tn in Caco-2) were increased. Western blots of cell homogenates showed differences between KATO III and Caco-2 in MUC 1 apomucin protein antigens, in sialyl Lex and in sialyl Tn antigens. We conclude that benzyl-alpha-GalNAc does inhibit the glycosylation of mucin in KATO III gastric cancer cells as in human colon cancer cells, but that alterations in mucin antigens occur in a cell line-specific manner.

Acetylgalactosamine↗

PubMed1995

Cystic fibrosis and pancreatic cancer cells synthesize and secrete MUC1 type mucin gene product.

The purpose of this study was to determine the biochemical and molecular characteristics of mucin synthesized by cystic fibrosis cells (CFPAC-1), a pancreatic cancer cell line derived from a patient with cystic fibrosis, and pancreatic cancer (SW-1990) cell lines. High molecular weight glycoproteins (HMG) were quantified by [3H]-glucosamine labeling and chromatography on sepharose CL-4B. Mucin gene expression was determined by using cDNA probes for 2 distinct intestinal mucins (MUC2 and MUC3) and one stomach mucin (MUC1). The specific mucin core epitopes were confirmed by immunoblots using antibodies that recognize T, Tn, sialosyl Tn, MUC1, MUC2, and MUC3. The results of these experiments demonstrate that CFPAC-1 cells contained 1.25 fold and 1.4 fold more HMG in the membrane and cytosolic fractions, however, secreted 4-fold more HMG into the medium compared to SW-1990 cells. The HMG of SW-1990 was found to be mucinous in nature and not proteoglycans, as it was not susceptible to hyalurinidase, heparinase and chondroitinase ABC. The HMG of CFPAC-1 was also predominantly (80%) mucinous but with small amounts of proteoglycans. mRNA and immunoblot analysis suggest that these CFPAC-1 and SW-1990 cells predominantly express MUC1 apomucin, small amounts of MUC2 apomucin, and no MUC3. Pulse chase labeling and immunoprecipitation of MUC1 type mucin using the 139H2 monoclonal antibody demonstrated that different sizes of mucin gene product were present in both cell lines, corresponding to the known length polymorphism of this mucin. Both T and Tn antigens were significantly higher in CFPAC-1 and SW-1990 cells as compared to sialosyl Tn antigen. These findings were associated with the increased activities of polypeptidyl N-acetylgalactosaminyl transferase and b1,3-galactosyltransferase. These investigations demonstrate for the first time that cystic fibrosis cells (CFPAC-1) secrete and synthesize high amounts of mucin which is associated with high levels of MUC1 mRNA, low levels of MUC2 mRNA and non detectable MUC3 mRNA.

Amino Acid Sequence↗

PubMed1995

Regression of LNCaP human prostate tumor xenografts in athymic nude mice by 13-cis-retinoic acid and androgen ablation.

The present study was designed to investigate the effects of 13-cis-retinoic acid (13-cis-RA) (100 micrograms/mouse/day) and androgen ablation (castration) alone and in combination on growth of a human prostatic carcinoma line (LNCaP) transplanted to athymic nude mice as an experimental model. The results of these studies suggest that; (1) androgen ablation (castration) significantly decreased the size of LNCaP xenograft as compared to untreated animals; (2) when 13-cis-RA was administered to nude mice carrying established tumors (0.51 +/- 0.04 cm3), the tumor size was significantly reduced as compared to untreated controls (0.65 +/- 0.06 cm3 versus 1.63 +/- 0.12 cm3). About 50% of the animals in this group showed xenografts necrosis followed by complete regression of tumors by five months; (3) the combination of androgen ablation and 13-cis-RA treatment to nude mice carrying tumors showed synergistic effect in decreasing the tumor size. These results indicate that combination therapies based on androgen ablation and retinoid administration may be a useful approach for the treatment of prostate cancer.

Animals↗

PubMed1995

Chronic penile denervation in the rat: effect on cavernous tissue morphology and function.

We evaluated the effects of chronic penile denervation on cavernous tissue morphology and function in 36 Sprague-Dawley rats. At age seven weeks, 18 animals underwent bilateral cavernous nerve neurectomy: 18 animals underwent sham operation as a control. A functional, biochemical and morphological assessment of the rats' penises was performed at 4 months. In denervated rats, intracavernous pressure failed to rise with electrostimulation of the pelvic plexus. However, a normal rise in pressure was found with direct intracavernous injection of sodium nitroprusside and papaverine. Sodium dodecylsulfate polyacrylamide (SDS) gel electrophoresis of the penile homogenate showed subtle differences between denervated and control animals. Based upon the histological findings there was no difference in staining of the cavernous tissue for acetylcholinesterase- and catecholamine-positive nerve fibers between experimental and control animals, since the innervation density was not quantified and the number of fibers was not counted. We conclude that chronic cavernous nerve neurectomy does not cause significant morphological or functional changes to the penile erectile tissue of rats.

Acetylcholinesterase↗

PubMed1994

Equilibrium linkage analysis of cardiac thin filament assembly. Implications for the regulation of muscle contraction.

A major focus in studies of muscle contraction has been the effect of Ca2+ on the interactions among the thin filament's five constituent polypeptides: actin, tropomyosin, troponin C (TnC), troponin T (TnT), and troponin I (TnI). We have investigated these interactions by analyzing thin filament assembly as a linear lattice binding problem with linkage relationships in the associations of tropomyosin, actin, and troponin. Binding of TnT, the binary TnT.TnI complex, or the ternary troponin complex (+/- Ca2+) to tropomyosin was measured spectrofluorimetrically after labeling cardiac tropomyosin with N-(1-pyrene)iodoacetamide. The affinity constants ranged between 0.2 and 0.6 microM-1 in the presence of 300 mM KCl. Also, the affinities of tropomyosin, tropomyosin-troponin, tropomyosin.TnT, and tropomyosin.TnT.TnI for an isolated site on F-actin were determined. The actin association constants were 0.0006 microM-1 for tropomyosin, 1 microM-1 for tropomyosin.TnT, 2 microM-1 for tropomyosin.TnT.TnI, 0.5 microM-1 for tropomyosin.troponin, and 0.5 microM-1 for tropomyosin-troponin.Ca2+. Linked equilibrium analysis permitted calculation of the affinities for actin.tropomyosin of TnT (400 microM-1), TnT.TnI (1600 microM-1), troponin (500 microM-1), and troponin.Ca2+ (300 microM-1). Therefore, both troponin and tropomyosin.troponin retain high actin-affinity even when Ca2+ is present or when TnI is removed, and even in the absence of cooperative contributions. The results are discussed in consideration of increasing evidence for a Ca(2+)-regulated azimuthal movement of tropomyosin on F-actin (Lehman, W., Craig, R., and Vibert, P. (1994) Nature 368, 65-67). It is proposed that tropomyosin movement may be due to switching between TnI-mediated and TnT-mediated binding of troponin-tropomyosin to distinct sites on F-actin.

Actins↗

PubMed1994

Inhibition of tumorigenic potential and prostate-specific antigen expression in LNCaP human prostate cancer cell line by 13-cis-retinoic acid.

Prostate-specific antigen (PSA) is a member of the kallikrein family and has been an important biological marker for prostate cancer. The mechanisms regulating PSA expression in prostatic cancer cells are unclear. The present study was designed to elucidate the role of 13-cis-retinoic acid (RA) in regulation of PSA and the tumorigenic potential of the human prostate cancer cell line LNCaP. The growth regulation of LNCaP cells was examined by DNA synthesis and doubling time. The tumorigenic potential of prostate cancer cells was analyzed by soft agar colony-forming assay, in vitro invasion assay, type IV collagenase assay and binding to extracellular matrix assay. The nuclear receptors for retinoic acid (RAR alpha, -beta, -gamma and RXR alpha, -beta, -gamma) as well as PSA mRNA were determined by Northern blot using specific oligonucleotide probes. Our results suggest that 13-cis-RA significantly inhibits PSA secretion and expression both at the mRNA and protein levels compared with untreated cells. Electron microscopic studies suggest that after 13-cis-RA treatment, cells become more differentiated as they contain lumina, lined by plasma membrane and microvilli. Prostate cancer cell growth and tumorigenic potential after 13-cis-RA treatment was significantly decreased compared with controls. Nude mice tumorigenicity studies showed that 13-cis-RA-treated cells produced significantly smaller tumors compared with untreated cell tumors. There was also a significant increase in the expression of RXRa mRNA after 13-cis-RA treatment compared with untreated cells.

Animals↗

PubMed1994

Stable differentiation of a human colon adenocarcinoma cell line by sodium butyrate is associated with multidrug resistance.

Colorectal cancers are often composed of cell types representing various differentiated cell lineages, however little is known concerning the relationship of differentiation and drug resistance in these cancers. The present study was performed to develop and characterize a stable, differentiated clone of the human colon cancer cell line LS174T and to characterize the drug resistance of this cell line in relation to its undifferentiated parental cell line. LS174T cell line was treated with the differentiating agent sodium butyrate (0.5 mM) for 30 days, then recultured in standard medium. Foci of flat-appearing cells appeared and were isolated using cloning rings, and subcloned. One subclone was designated LS174T-D. The LS174T-D clone maintains a stable, differentiated phenotype in standard culture conditions in the absence of sodium butyrate. It is characterized by the formation of a polarized monolayer with dome formation and the presence of prominent apical microvilli and tight junctions. This cell line demonstrated reduced growth in soft agar and nude mice compared with the parental cell line. LS174T-D cells expressed immunoreactive intestinal mucin antigens and brush border enzymes dipeptidyl aminopeptidase (DAP)-IV and aminopeptidase. The activities of DAP-IV and aminopeptidase were increased 5.6-fold and 3.4-fold, respectively, in LS174T-D compared with parental cells. Proliferation assays demonstrated that, compared with the parental cell line, LS174T-D cells were more resistant to doxorubicin (93-fold), cisplatin (23-fold), 5-fluorouracil (12-fold), 5-fluorodeoxyuridine (31-fold), and methotrexate (12.5-fold). Intracellular uptake of (3H)-5-fluorodeoxyuridine did not differ significantly in the differentiated and undifferentiated cell lines. Levels of mdr-1 p-glycoprotein measured by Western blot and RNA Northern blot assays were also similarly low in both cell lines. However, total glutathione content and glutathione-S-transferase activities were increased in LS174T-D cells by sixfold and threefold, respectively, compared with parental cells. Depletion of glutathione by pretreatment with DL-buthionine sulfoximine reversed LS174T-D resistance to cisplatin. Long-term treatment with sodium butyrate induces or selects for colon cancer cells with features of enterocytic differentiation. This stably differentiated cell line is associated with glutathione-mediated multidrug resistance, and provides a model for further studies of differentiation in normal and cancerous colon.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

13-cis-retinoic acid-mediated growth inhibition of DU-145 human prostate cancer cells.

The purpose of this study was to examine the effects of 13-cis-retinoic acid (13-cis-RA) on the growth regulation of DU-145 human prostatic cancer cells. The results of these experiments demonstrate that cell growth and metastatic potential of DU-145 cells were significantly inhibited by 13-cis-RA (10 microM). In order to elucidate the possible molecular mechanisms of 13-cis-RA action on prostate cancer cells, we examined the expression of nuclear receptor genes (hRXR alpha) and found that 13-cis-RA treated cells showed higher mRNA expression for hRXR alpha nuclear receptors compared to untreated cells. To elucidate further the possible biochemical mechanisms associated with these alterations, we analyzed the phosphorous metabolites by MR spectroscopy and found that the major metabolites were PME, (PC, PE) and DPDE (UDP-GalNAc, UDP-GLcNAc). The DU-145 cells and xenografts, which were both treated with 13-cis-RA, showed a two-fold decrease in DPDE's, compared to their controls. The higher resolution spectra of perfused cells revealed that phosphocholine levels were twice as high in 13-cis-RA-treated DU-145 cells as compared to untreated cells. These investigations demonstrate for the first time that 13-cis-RA inhibits the growth of human prostatic cancer cells, and this inhibition is associated with an increase in hRXR alpha nuclear receptor gene expression and alterations in phosphorous metabolites detected by 31P MR spectroscopy.

Animals↗

PubMed1993

Mucin synthesis and secretion in various human epithelial cancer cell lines that express the MUC-1 mucin gene.

Previous studies have suggested that mucin gene expression is tissue-specific; however, the relationship between unique mucin gene products and the biochemical properties of mucins is unknown. The purpose of this study was to determine the biochemical and molecular characteristics of mucin synthesized by adenocarcinoma cell lines derived from breast (ZR-75-1), stomach (MGC-803), pancreas (Capan-2), and lung (Chago K-1). Mucin was quantitated by [3H]glucosamine labeling and Sepharose CL-4B chromatography. The mucinous nature of the labeled high molecular weight glycoproteins (HMG) was verified by alkaline borohydride treatment, cesium chloride density gradient ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific mucin gene expression was determined using cDNA probes for 2 distinct intestinal mucins (MUC-2 and MUC-3) and one breast cancer mucin (MUC-1). Specific core mucin proteins were confirmed by immunoblots using antibodies that recognize MUC-1, MUC-2, and MUC-3 core peptides. These experiments demonstrate that all cell lines contained HMG in the medium, cytosol, and membrane fractions. The HMG was mucinous in breast, pancreatic, and lung cell lines. In contrast, most of the HMG secreted by the gastric cell line was proteoglycan-like, due to its susceptibility to hyaluronidase, heparinase, and chondroitinase avidin-biotin complex. Ion-exchange (DEAE-Sephacel) chromatography of [3H]glucosamine-labeled HMG demonstrated that the acidic or basic nature of the mucin was different in all cancer cell lines tested. Despite these differences, mRNA and immunoblot analysis suggest that all cell lines predominantly express MUC-1 apomucin, small amounts of MUC-2 apomucin, and no MUC-3. Immunoprecipitation of MUC-1-type mucin using the 139H2 monoclonal antibody demonstrated that different sizes of mucin peptides were present in all cell lines, corresponding to the known length polymorphism of this mucin. The amount and nature of carbohydrate epitopes were analyzed by immunoblots using anti-T (peanut lectin), anti-Tn (91S8 monoclonal antibody), and anti-sialosyl Tn (JT10e monoclonal antibody). T and Tn antigens were significantly higher in breast and pancreatic cells as compared with lung and gastric cell lines. These findings correlated with increased activities of polypeptidyl N-acetylgalactosaminyl transferase and beta-1,3-galactosyltransferase.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗

PubMed1993

Heterogeneity of mucin gene expression in normal and neoplastic tissues.

To determine the relative expression of distinct mucin genes in normal and neoplastic tissue, antibodies and cDNA probes that recognize the core tandem repeat sequences of membrane-bound (MUC1) and secreted (MUC2 and MUC3) mucins were used for immunohistochemical and RNA Northern and slot-blot analysis. MUC1 mRNA was detected in all epithelial tissues tested. MUC1 core peptide, recognized by monoclonal antibodies 139H2 and DF3, was highly expressed on apical membranes of bronchus, breast, salivary gland, pancreas, prostate, and uterus, and was sparsely expressed in gastric surface cells, gallbladder, small intestine, and colonic epithelium. In contrast, MUC2 and MUC3 gene expression was primarily restricted to the intestinal tract. MUC2 mRNA was highly expressed in normal jejunum, ileum, and colon, compared with very low levels in normal bronchus and gallbladder. MUC3 mRNA was highly expressed in normal jejunum, ileum, colon, and gallbladder. Immunohistochemical studies using antibodies against synthetic MUC2 (anti-MRP) and MUC3 (anti-M3P) peptides indicate that MUC2- and MUC3-producing cells in the gastrointestinal tract are distinct. Goblet cells of the small intestine and colon reacted strongly with anti-MRP, whereas M3P reactivity was restricted to columnar cells of small intestinal villi, surface colonic epithelium, and gallbladder. Mucin protein epitopes and mRNA levels were frequently altered in adenocarcinomas compared to corresponding normal tissues. Alterations included increased expression, aberrant expression, and, less frequently, loss of expression. Increased MUC1 immunoreactivity was observed in most adenocarcinomas of the breast, lung, stomach, pancreas, prostate, and ovary. In addition, with the exception of prostate cancer, focal aberrant expression of MUC2 and MUC3 epitopes was frequently observed. Increased MUC1, MUC2, and MUC3 epitopes were present in colon adenocarcinomas of all histological subtypes, with the greatest increase of MUC2 epitopes observed in colloid (mucinous) colon cancers. MUC2 or MUC3 mRNA levels were increased in colloid colon cancer compared with normal colon, however in well- and moderately well-differentiated colon cancers MUC1, 2 and 3 mRNA levels were decreased. Compared with corresponding normal tissue, MUC1 mRNA levels were increased in breast cancer and well-differentiated lung cancers, and MUC3 mRNA was increased in gastric adenocarcinomas. Normal stomach lacked both MUC2 and MUC3 immunoreactivity and mRNA, however, MUC2 and MUC3 proteins and mRNA were highly expressed in gastric intestinal metaplasia. In conclusion, mucin genes are independently regulated and their expression is organ- and cell type-specific. Furthermore, neoplastic transformation is associated with dys-regulated expression of both membrane-bound and secreted mucin core protein epitopes and may be due to altered mucin mRNA levels and/or altered mucin glycosylation.

Adenocarcinoma↗

PubMed1993

Citrate alterations in primary and metastatic human prostatic adenocarcinomas: 1H magnetic resonance spectroscopy and biochemical study.

The objectives of this study were to quantitatively verify the low levels of citrate previously observed in primary human prostatic adenocarcinomas and to determine whether citrate was further reduced in metastatic prostatic cancer. This was accomplished by comparison of citrate concentrations of DU 145 xenografts (a poorly differentiated human prostatic adenocarcinoma cell line grown in nude mice) with concentrations in primary human adenocarcinomas. Following in vivo 1H NMR studies of DU 145 xenografts, citrate concentrations of DU 145 xenografts and surgically removed primary prostatic adenocarcinoma tissue were determined by quantitative high resolution 1H NMR and enzymatic assay. The most significant findings of this study were that citrate concentrations in primary human adenocarcinomas (3.74 +/- 0.19 mumol/g wet weight) were significantly lower than those observed for normal and benign hyperplastic (BPH) prostatic tissues. Furthermore there was a further ten-fold reduction of citrate associated with DU 145 xenografts (0.31 +/- 0.028 mumole/g wet weight) compared with primary prostatic cancer. DU 145 xenografts also exhibited higher levels of uridine diphosphosugars and choline containing metabolites relative to primary prostatic adenocarcinomas. These findings support the hypothesis that citrate is low in primary prostatic cancer and further reduced in metastatic disease.

Adenocarcinoma↗

PubMed1993

Effect of glucose and confluency on phosphorus metabolites of perfused human prostatic adenocarcinoma cells as determined by 31P MRS.

A series of perfused cell 31P MRS studies were conducted using a well established human prostate adenocarcinoma cell line (DU 145) at different phases of growth, and exposed to varying glucose concentrations during growth. The spectral characteristics of perfused DU 145 cells were compared with the same cells grown in nude mice (xenografts). Perfused DU 145 cells had lower levels of inorganic phosphate and phosphocreative relative to in vivo nude mice xenografts. 31P MR spectra obtained from perfused cells at different phases of growth and exposed to varying glucose concentrations during grown suggest that increases in diphosphodiester levels are associated with high glucose concentrations and confluency. Perfused DU 145 cells grown in 5.5 mM glucose and harvested at log phase of growth best reflected the phosphorus MR spectra of the same cell line grown in nude mice.

Adenocarcinoma↗