Biotransformation of biphenyl by Paecilomyces lilacinus and characterization of ring cleavage products.

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4'-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.

Monoclonal antibody Adnab-9 defines a preneoplastic marker in epithelium at risk for adenocarcinoma of the small intestine.

Unlike colorectal cancer, risk markers for adenocarcinoma of the small intestine (ASI) have not been identified. Because the demographic and pathological features of both of these diseases are similar, immunohistochemistry was performed using monoclonal antibodies for three colonic premalignant markers, Adnab-9 (recognizes a colonic adenoma epitope), CaCo3/61, and FBB2/29 (small intestine proteoglycans expressed ectopically in colonic neoplasms), in normal and neoplastic small intestinal epithelium, and the results were compared with normal controls. Adnab-9 was also examined in 20 familial adenomatous polyposis (FAP) patients, a population known to be at an increased risk for ASI. Immunohistochemistry in normal and neoplastic tissue (adenoma, adenocarcinoma) from 18 patients with primary adenocarcinoma of the small intestine was compared with normal small intestine from 10 nonneoplastic controls. Four of 10 (40%) cases of normal small intestinal epithelium from controls were mildly positive in less than 10% of crypts, versus strong staining (>50% of crypts) in 16 of 18 (89%) patients with adenocarcinoma, and in 17 of 20 (85%) patients with FAP (P<.05). Adnab-9 predominantly stained Paneth cells as well as rare crypt and basal villous goblet cells. Adenomatous epithelium from the adenocarcinoma cases and adenomas from the FAP patients showed staining of Adnab-9 in 63% and 78% of cases, respectively. Only 17% of adenocarcinomas were positive for Adnab-9. In contrast, neither CaCo3/61 nor FBB2/29 showed any significant differences in the degree of staining in normal small intestinal epithelium in patients with adenocarcinoma compared with controls. Enhanced Adnab-9 staining in normal small intestinal epithelium from patients who harbor adenocarcinoma, and in FAP patients, supports its role as a risk marker of small intestinal neoplasia.

Effect of spermine administration on pancreatic maturation in unweaned rats.

The effect of oral administration of spermine on pancreatic maturation was investigated in the suckling rat. The treatment consisted of 0.3-0.4 mmol spermine kg-1 body weight given orally once a day for 3 days starting at day 11 after birth. Spermine administration does not adversely affect the growth of the pancreas (wet weight, protein and DNA contents remain unchanged). The proliferating cell nuclear antigen (PCNA) index decreases significantly in spermine-treated rats, indicating that spermine slows down the proliferation rate of the organ. The enzymatic activities of trypsin, chymotrypsin and alpha-amylase are increased significantly in the pancreas of spermine-treated rats. The morphology of the organ seems affected as shown by hematoxylin-eosin staining: a cytoplasm indicative of higher synthetic activity is visible after spermine treatment. We conclude that spermine treatment of unweaned rats can induce precocious biochemical and morphological maturation of the exocrine pancreas, pushing the organ forward in the process of differentiation (closer to the adult stage).

Early weaning induces jejunal ornithine decarboxylase and cell proliferation in neonatal rats.

Increased ornithine decarboxylase (ODC) activity is associated with rapid cell proliferation in many cell types. The cellular effects of early weaning on intestinal development are not well established. To investigate whether ODC is involved in intestinal growth after early weaning, we precociously weaned suckling rats on postnatal d 15 and followed through d 21 (6 d after early weaning). Age-matched suckling pups served as controls. Rat pups were killed 1, 2, 3 and 6 d after early weaning and jejunal mucosa was assayed for ODC and sucrase activities, and protein and DNA contents. Jejunal cell proliferation was monitored by bromodeoxyuridine immunohistochemistry. Elevated jejunal ODC activity 1 d after early weaning was the earliest cellular event that was detected in the current study. ODC activity peaked at d 3 (about 15-fold greater than age-matched unweaned suckling controls). Sucrase activity was elevated at d 2 after weaning and peaked at d 3 (about 10-fold greater than controls). Greater bromodeoxyuridine immunostaining in early weaned rats occurred on d 3. Protein and DNA contents were greater in jejunal mucosa of early weaned rats at d 6. Serum corticosterone levels were elevated on d 1 and d 2 after early weaning compared to controls. To explore whether the intake of nonpurified diet played a role, we also compared the induction of jejunal ODC activity in early weaned pups and pups that were food-deprived for 1 d. ODC activity was not greater in the food-deprived group compared to suckling controls while the early weaned group had 6-fold greater activity 1 d after early weaning. Early weaning stimulates jejunal cell proliferation and differentiation. The temporal sequence of increased ODC activity followed by increases in other growth variables suggests that the induction of ODC activity may act as an early marker of intestinal growth during early weaning.

Neuropeptide Y (NPY) enhances proliferation of human colonic lamina propria lymphocytes.

Neuropeptide Y (NPY) is one member of a family of peptides with a wide range of physiological effects on the CNS, cardiovascular, and respiratory systems. NPY is widely distributed throughout the peripheral and central nervous systems. It has also been found within the colon, liver and gallbladder in close anatomic proximity to the mucosal immune system. In this study, we investigated the effect of NPY on human gut mucosal immune function. We examined colonic lamina propria lymphocyte (LPL) proliferation by measuring DNA synthesis, ornithine decarboxylase (ODC) activity, and polyamine biosynthesis. NPY enhanced ODC activity and polyamine biosynthesis in Con A-stimulated LPL, and enhanced thymidine incorporation into Con A-stimulated LPL but not into monocyte-depleted LPL. Moreover, exogenous IL1-beta partially restored NPY's stimulatory effect on monocyte-depleted LPL DNA synthesis. Our results demonstrate that NPY enhances human colonic LPL proliferation and that this effect is partially IL1-beta dependent. Our data also suggest that NPY's effect may be mediated via polyamine biosynthesis. We postulate that the NPY may have an important impact on human mucosal immune function.

Analysis of ODC and c-myc gene expression in hepatocellular carcinoma by in situ hybridization and immunohistochemistry.

We quantitated mRNA and protein for ornithine decarboxylase (ODC) and c-myc in formalin-fixed liver sections from 25 specimens of hepatocellular carcinoma (HCC) and seven normal livers by a non-radiolabeled in situ hybridization technique and immunohistochemistry. This non-radioactive in situ hybridization technique was highly specific, with virtually no background, and permitted quantitative analysis based on optical density. Reaction products were quantitated with computer-assisted microdensitometry. Samples were classified as normal, adjacent uninvolved, cirrhosis, well-differentiated HCC, and poorly-differentiated HCC. There was a progressive increase in all four parameters measured, ODC mRNA and protein, and c-myc mRNA and protein, from normal, to adjacent uninvolved liver, to cirrhosis, to well-differentiated HCC, to poorly-differentiated HCC. The sole exception was that ODC mRNA was lowest in cirrhosis. The patterns of ODC and c-myc gene expression are similar in HCC. The quantitative detection of ODC mRNA, c-myc mRNA, and their protein products in hepatocellular carcinoma and cirrhosis by in situ hybridization and immunohistochemical techniques may have a potential role in the study of hepatocarcinogenesis and in the diagnosis of hepatocellular carcinoma.

A defined collagen substrate and nutrient diffusion gradient culture system allows human colon cancer cells to assume a more differentiated phenotype.

A culture system is presented in which a biologically more relevant substrate in the form of a collagen membrane and a biologically more relevant diffusion gradient system for nutrient delivery to the cells are provided. Five established human colon cancer cell lines (Caco-2, DLD-1, Widr, HCT 116 and HCT 8) were cultured in this system and three of them showed increased differentiation compared with the same cells cultured on the usual cell culture substrates of plastic and glass and with cells grown in an anchorage-independent manner. Caco-2 cells grow on plastic as a monolayer of large pleomorphic cells with scant mucin production. When cultured in a gradient diffusion system in a sandwich of type I/IV collagen the Caco-2 cells showed the highest degree of morphological and biochemical differentiation as evidenced by cellular alignment, glandular formation and mucin production. Similarly DLD-1 cells exhibited the greatest degree of morphological and biochemical differentiation in a gradient system in a type I/IV collagen sandwich. HCT 116 and HCT 8 cells, however, showed little change in differentiation phenotype under any of the 11 culture conditions tested. Widr cells grew in a type I/IV collagen sandwich in a gradient diffusion system as multilayered sheets of cells with intercellular spaces, suggestive of a moderate increase in differentiation phenotype. As judged by morphology and mucin production a range of differentiation capacities is exhibited by the five cell lines tested under the optimal differentiating culture conditions, with the order from most able to differentiate to least able to differentiate being Caco-2 > or = DLD-1 > or = Widr > HCT 116 > or = HCT 8.

ODC activity and polyamine levels in isolated human colonocytes.

Ornithine decarboxylase (ODC) is the first and often rate-limiting enzyme in polyamine biosynthesis. ODC and polyamines (putrescine, spermidine, spermine, and cadaverine) have an essential role in cell proliferation. In this study, we investigated ODC activity and the polyamine levels of normal human colonocytes isolated from the upper and lower crypt regions. We found no significant differences in ODC activity between upper and lower crypt regions (mean +/- SEM: 105 +/- 60 and 103 +/- 52 pmol CO2/mg protein/hr, respectively). This result was further substantiated by ODC immunoreactive antibody staining technique. Levels of polyamines (putrescine, spermidine, spermine, and cadaverine) were similar in the upper and lower crypt regions (mean +/- SEM; upper/lower: 79 +/- 29/79 +/- 18; 189 +/- 116/ 137 +/- 38; 174 +/- 58/204 +/- 35; and 52 +/- 10/51 +/- 10 nmol/mg protein, respectively). Acetyl-polyamines (acetyl-putrescine, acetyl-spermidine, and acetyl-spermine) levels in human colonocytes showed no significant differences between upper and lower crypt regions (mean +/- SEM; U/L: 368 +/- 109/408 +/- 89, 63 +/- 22/51 +/- 12, and 39 +/- 12/41 +/- 14 nmol/mg protein, respectively). Our results suggest that in isolated normal human colonocytes, ODC activity and polyamine levels are similar in the upper and the lower crypt regions.

Cellular distribution of a colonic adenoma-associated antigen as defined by monoclonal antibody Adnab-9.

Adenomatous colonic polyps constitute a precursor for colorectal cancer. Antibodies to these precancerous lesions might identify specific early tumor antigens. Adnab-9 is a murine monoclonal antibody raised against membranes of colonic adenomas. Adnab-9 binding in colonic washings (effluent) correlates with the presence of colorectal cancer. Immunohistochemical staining with Adnab-9 shows cytoplasmic reactivity in scattered cells in 4 of 31 adenomatous tissue sections, 0 of 14 sections of colorectal cancer cells, and 1 of 8 normal-appearing colonic mucosa specimens examined. Adnab-9 recognized a dominant M(r) 87,000 protein species in tissue extracts in the membrane-bound fraction of effluent by Western blotting. Adnab-9 binding by enzyme-linked immunosorbent assay in adenomatous extracts is higher than cancer or normal tissue, is membrane-bound, and is absent from established colorectal cancer cell lines. This distribution and nature of immunostaining suggest that Adnab-9 recognizes a determinant associated with the membrane component of a subpopulation of adenoma cells which may have a role in early colorectal neoplasia.

Detection of ornithine decarboxylase messenger RNA in human hepatocellular carcinoma by in situ hybridization.

Ornithine decarboxylase (ODC) has been shown by biochemical analysis, to be important for cell proliferation and carcinogenesis in a variety of tissues, including the liver. We detected messenger RNA (mRNA) specific for the enzyme ODC in 18 patients with hepatocellular carcinoma by an in situ hybridization technique using a radiolabelled ODC probe on formalin-fixed liver specimens. Adjacent uninvolved liver tissues were used as controls. Among the adjacent uninvolved liver tissues, five showed evidence of cirrhosis. Poorly differentiated hepatocellular carcinoma has significantly higher levels of ODC mRNA than does well-differentiated hepatocellular carcinoma, which in turn has a significantly higher ODC mRNA level than adjacent uninvolved liver tissues; tissues showing evidence of cirrhosis, on the other hand, had a significantly lower ODC mRNA level than adjacent uninvolved liver tissue. This pattern of ODC gene expression in hepatocellular carcinoma is similar to the pattern of expression of other oncogenes in liver tumours. The quantitative detection of ODC mRNA in hepatocellular carcinoma by in situ hybridization may help elucidate the potential role of ODC in hepatocarcinogenesis.

v-Ha-ras oncogene insertion: a model for tumor progression of human small cell lung cancer.

Small cell lung cancer (SCLC) manifests a range of phenotypes in culture that may be important in understanding its relationship to non-SCLCs and to tumor progression events in patients. Most SCLC-derived cell lines, termed "classic" SCLC lines, have properties similar to SCLC tumors in patients, including high expression of neuroendocrine markers and low c-myc oncogene expression. A significant number of SCLC lines characterized as "biochemical or morphologic variant" SCLC lines have decreased levels of endocrine differentiation markers associated with increased proliferative indices, amplification of the c-myc oncogene, and growth patterns and biochemical markers more typical of non-SCLCs. To delineate further the relationships between these phenotypes and the molecular events involved, we have inserted the v-Ha-ras gene in SCLC cell lines with (biochemical variant) and without (classic) an amplified c-myc gene. These two SCLC subtypes had markedly different phenotypic responses to similar levels of expression of v-Ha-ras RNA. No biochemical or morphologic changes were observed in classic SCLC cells. In contrast, in biochemical variant SCLC cells, v-Ha-ras expression induced features typical of large cell undifferentiated lung carcinoma, including adherent monolayer growth patterns, increased cloning efficiency, increased levels of non-SCLC cell markers, ultrastructural characteristics and an acquired resistance to polyamine depletion typical of large cell carcinoma, but not SCLC, in vitro. Expression of v-Ha-ras in biochemical variant SCLC cells directly demonstrates that important transitions can occur between phenotypes of human lung cancer cells and that these may play a critical role in tumor progression events in patients. The findings provide a model system to study molecular events involved in tumor progression steps within a series of related tumor types.

Intracellular growth factors in polycythemia vera and other myeloproliferative disorders.

In polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis, hematopoietic cell proliferation is increased in the absence of a recognizable stimulus, suggesting the autonomous production of growth factors in these disorders. Sonicates of peripheral blood mononuclear cells (PBMNC) from patients with polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis contained soluble factors that stimulated the proliferation of quiescent-confluent 3T3 cells. PBMNC sonicates from normal individuals; from patients with secondary erythrocytosis, chronic myelogenous leukemia, B-cell chronic lymphocytic leukemia, and acute myelogenous leukemia; and from K-562 and HL-60 cells did not stimulate proliferation. Polycythemia vera PBMNC sonicates also induced anchorage-independent colony formation in soft agar by normal rat kidney fibroblasts. Both the mitogenic and transforming activities of the polycythemia vera PBMNC sonicates resided in the T-lymphocyte-depleted mononuclear fraction of the PBMNC and were not secreted. By gel filtration, reversed-phase HPLC and NaDodSO4/PAGE, the mitogenic and transforming activities in the polycythemia vera PBMNC were localized to three proteins with molecular masses of 13-, 17-, and 65-kDa. The 13-kDa protein was only mitogenic, and the 17-kDa protein was only transforming, whereas the 65-kDa protein had both mitogenic and transforming activity. These proteins may be involved in the autonomous hematopoiesis that characterizes polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis.

Human medullary thyroid carcinoma in culture provides a model relating growth dynamics, endocrine cell differentiation, and tumor progression.

We used an unique model, human medullary thyroid carcinoma (MTC) in culture (the TT line), to study features of neuroendocrine-related biochemistry in relationship to growth, differentiation, and tumor progression. Tumor tissues from patients with virulent MTC contain a very heterogeneous distribution of cells staining for calcitonin (CT) and have a high ratio of intracellular L-dopa decarboxylase activity (DDC) to CT. We found, in a culture line of MTC derived from a patient with virulent disease, that the degree of the inverse relationship between DDC and CT and the heterogeneous cellular distribution of CT probably relate to the rate of cellular growth and the biochemical set of individual cell clones. During exponential growth of the parent TT cell line, intracellular DDC and CT varied. DDC increased by 70% and CT decreased by 40%. Single time-point measurements in 54 cell clones or highly enriched cell populations revealed a more dramatic variability for CT (15-fold) than for DDC (5-fold). During growth of the clones having the highest and lowest CT measurements, respectively, inverse dynamics between DDC and CT were again found. However, each clone maintained a distinct range of CT during the entire growth curve, with a 2- to 4-fold difference in CT between the two clones throughout. In the low producing CT clone, ratios between DDC and CT rose to greater than 1.0 during growth, a very high value found before this study only in MTC tissues from patients with virulent disease. Immunohistochemical staining for CT of parent cells and clones grown on embryonic chick skin revealed increased cellular heterogeneity for CT distribution during growth. The TT line provides a powerful tool to study neuroendocrine related biochemical events in relationship to growth, differentiation, and tumor progression in MTC. Our in vitro findings in the TT line well explain observations made previously in patients. We conclude that: (1) DDC, a neural property of MTC, is an early differentiation marker as compared to CT and that the differentiation status of MTC cells varies inversely with cell growth rate; and (2) in patients with MTC, the virulence of the tumor probably varies inversely with differentiation status. The inverse ratio of DDC to CT is probably determined in MTC by the proportion of rapidly growing cells and numbers of cell clones which have a poor ability for maturation.

Retinoic acid binding protein in human and experimental pancreatic carcinomas in hamsters.

Cytoplasmic retinoic acid binding protein (cRABP) is present in human fetal pancreas and becomes nondetectable in the normal adult pancreas. The binding protein for retinoic acid becomes apparent again in pancreatic cancer. Similar fluctuations in the content of cRABP occur in the hamster. The binding protein is undetectable in the normal adult hamster pancreas, while it was detected in several transplantable adenocarcinomas in the Syrian golden hamster.

Retinoic acid binding protein in normal and neopolastic rat prostate.

Sucrose density gradient analysis of cytosol from normal and neoplastic rat prostatic tissues exhibited a peak of (3H) retinoic acid binding in the 2S region, corresponding to the cytoplasmic retinoic acid binding protein (cRABP). In the Fisher-Copenhagen F1 rat, cRABP was present in the lateral lobe, but could not be detected in the ventral nor in the dorsal prostatic lobes. Four sublines of the R-3327 rat prostatic tumor contained similar levels of this binding protein. The absence of cRABP in the normal tissue of origin of the R-3327 tumor, the rat dorsal prostate, and reappearance in the neoplastic tissues follows a pattern described in other human and animal tumors. The occurrence of cRABP in the well-differentiated as well as in the anaplastic R-3327 tumors in which markers which reflect a state of differentiation and hormonal regulation, such as androgen receptor, 5 alpha reductase, and secretory acid phosphatase are either markedly reduced or absent, points to cRABP as a marker of malignant transformation.

Decrease in rat uterine estrogen receptors during aging: physio- and immunochemical properties.

Rat uterine estrogen receptors decrease in concentration by about 45% between maturity (6-12 months) and senescence (22-24 months). No change in binding affinity occurs over this period. No age differences were observed in the thermostability, hormone-induced stability, or specificity of binding for various steroids. Most importantly, immunochemical titration of receptors with specific antiserum revealed that the ratio of immunoreactive to functional receptor is not changed with age. Thus, the apparent loss of rat uterine estrogen receptors during aging does not seem to be due to the appearance of a nonfunctional receptor population detectable by presently available immunoreagents.

The effects of retinoids on proliferative capacities and macromolecular synthesis in human breast cancer MCF-7 cells.

The effects of various retinoids on the proliferative capacities and on the synthesis of DNA, RNA, and protein have been investigated in MCF-7 mammary carcinoma cells in culture. Of the various retinoids tested, retinoic acid revealed maximum activity in inhibiting cell proliferation and thymidine incorporation. The degree of inhibition of cell proliferation by the various retinoids paralleled their capacity to inhibit thymidine incorporation, suggesting suppression of DNA synthesis as a primary cause of restriction of cell growth by these compounds. Two nonepithelial human cell lines were tested for sensitivity to retinoids, and showed diminished responses compared with MCF-7 cells. This suggests a correlation between the ability of retinoids to exert control of differentiation and cell proliferation for a given cell type. Reversibility of the effect of retinoid treatment, high cell viability, and lack of retinoid-induced lysosomal enzyme release, as shown in our studies, indicate that cytotoxicity may be excluded as a cause of decreased cell proliferation and inhibition of thymidine incorporation by retinoids.

Effects of indomethacin on renomedullary interstitial cells.

The effects of indomethacin, an inhibitor of prostaglandin synthesis, on rat renomedullary interstitial cells were studied. Indomethacin, 5 mg. per kg. intravenously in divided doses over 48 hours, resulted in reduced granularity of interstitial cells (5.56+/-1.37 versus 9.85+/-1.07 granules per cell, (p is less than 0.001) and, at the same time, inhibited the incorporation of 14C-arachidonate into renomedullary phospholipids (715 + 11 versus 1299 + 42 c.pm.per mug. of lipid orthophosphate; p is less than 0.001) 14C-arachidonate incorporation into cortical phospholipids was not affected by indomethacin. There was a close correlation between individual granule counts and 14C-arachidonate incorporation into medullary phospholipids for both control (r=0.85) and indomethacin-treated animals (r=0.9). Radioactivity in the cytosol fraction was depressed by indomethacin reflecting inhibition of prostaglandin synthesis; cytosol radioactivity correlated closely with individual granule count (r=0.81) in the indomethacin-treated but not in the control rats. Indomethacin given as a single dose 4 hours prior to sacrifice resulted in a significant depression of 14C-arachidonate incorporation but did not affect granularity of interstitial cells. The results suggest that granularity of renomedullary interstitial cells reflects the activity of prostaglandin synthesis and the rate of prostaglandin precursor delivery from microsomal phospholipids.