Cyclic adenosine 3',5'-monophosphate during glucose repression in the rat liver.

Intragastric administration of glucose inhibits the induction of serine dehydratase and tyrosine aminotransferase by glucagon in rat liver, but has no effect on the increase in hepatic adenosine 3',5'-monophosphate resulting from administration of glucagon. Thus, glucose repression in mammalian liver, unlike catabolite repression in microorganisms, appears to operate independently of the amounts of cyclic nucleotide in the cells.

Induction of enzymes by glucagon, glucose repression, adenosine 3',5'-monophosphate concentration during carcinogenesis and in Morris 6918A hepatoma.

We have studied glucagon induction of enzymes, adenosine 3', 5'-monophosphate concentrations, and glucose repression in Morris 9618A hepatoma and in the liver of rats fed, for periods of up to 5 weeks, a solid diet containing 2-acetylaminofluorene or 3'-methyl-4-dimethylaminoazobenzene. While the basal levels of the enzymes serine dehydratase and tyrosine aminotransferase were the same as those found in control rats, their response to glucagon was reduced in experimental animals with or without tumors. However, the basal or glucagon-stimulated levels of adenosine 3', 5'-monophosphate in the liver of rats given the carcinogens were not changed. In Morris 9618A hepatoma, these parameters were, likewise, comparable to those in control animals. When glucose was administered to carcinogen-treated or tumor-bearing rats that had received a single dose of glucagon, there was no suppression of the increase in activity of serine dehydratase and tyrosine aminotransferase observed after glucagon treatment alone. The loss of glucose repression was seen already at 2 to 3 weeks following initiation of the carcinogenic diets. As previous studies had established for normal liver, the hormone-induced high levels of adenosine 3',5'-monophosphate remained unchanged also in Morris 9618A hepatoma and in rats given carcinogen. These results indicate that alterations in enzyme induction during chemical carcinogenesis are not the consequence of changes in adenosine 3',5'-monophosphate levels caused by carcinogens. The early disappearance of the glucose effect, which persists in slow-growing hepatomas, may be an expression of interference by carcinogens with the translation apparatus of the hepatic cell.

Effects of a high-sucrose diet on the development of enzyme-altered foci in chemical hepatocarcinogenesis in rats.

Dietary factors can modify metabolic events involved in the initiation, promotion, or progression of tumors. To determine whether a high-sucrose diet has any effect on the development of enzyme-altered foci during the promotion step of chemical hepatocarcinogenesis in rats, 1-day-old Sprague-Dawley rats were given a single i.p. dose of diethylnitrosamine; controls received an equivalent i.p. volume of 0.9% NaCl solution. At 21 days of age, the rats were weaned, segregated by sex, separated in groups, and fed modified AIN76A diets containing either 65% glucose or 65% sucrose, with or without 0.05% phenobarbital. At the end of a 4-week treatment period, the sucrose-fed control rats of either sex had significantly heavier livers than did those on the glucose diet. Enlarged livers were found also in the sucrose-fed diethylnitrosamine-treated female rats, which developed twice as many gamma-glutamyltranspeptidase-positive foci per sq cm of liver section than did those on the glucose diet. Addition of phenobarbital augmented the number of foci 3-fold in the sucrose group and 5-fold in the glucose group. Focus count per sq cm was similar in animals on the two phenobarbital-supplemented diets. Despite the absence of statistically significant liver enlargement, results analogous to those in females were obtained in carcinogen-treated males. Differences between treatments, however, were smaller. In both female and male rats, the DNA-synthesizing activity of hepatocytes in enzyme-altered foci was significantly higher than in the surrounding normal parenchymal cells, as determined by autoradiography. These studies indicate that a high-sucrose diet has a promoting effect during hepatocarcinogenesis induced in the rat by diethylnitrosamine and that this effect is weaker than that of 0.05% phenobarbital.

Nuclear DNA content of altered hepatic foci in a rat liver carcinogenesis model.

In individual altered hepatic foci (AHF), aneuploidy occurs before malignant changes can be diagnosed histologically (O. Sudilovsky and T. K. Hei. Fed. Proc., 42:2225, 1983). In the current experiments Sprague-Dawley rats of both sexes were given i.p. injections of diethylnitrosamine (50 mg/kg body weight) 18 h after partial hepatectomy and were given a choline-sufficient diet (CS) for 1 wk. Four treatment groups were then formed and fed CS, CS containing 0.05% phenobarbital (PHB), choline-deficient diet (CD), and CD with 0.05% PHB. An extra female group received infusions of saline after the hepatectomy and fared CD. Control animals were partially hepatectomized, inoculated i.p. with saline, and placed on CS. The rats were sacrificed 16 wk later, liver sections were stained with a combined Feulgen-gamma-glutamyl transpeptidase stain, and the DNA content of gamma-glutamyl transpeptidase-positive foci was measured cytospectrophotometrically. There were no AHF in the control animals. Hepatocytes from control livers and cells adjacent to foci in treated livers had peaks corresponding to the 2C, 4C, and 8C range. In AHF the ploidy, however, was predominantly diploid, tetraploid, or heterogeneous. The ratio of diploid to tetraploid cells in foci of rats provided with CS + PHB was 5.5 and in those supplied with CD + PHB was 0.09. This suggested that dietary manipulations change the nuclear DNA distribution of AHF. Aneuploidy was also present, as expected, in 4 of 33 AHF in the animals placed on CD + PHB. It was observed as well in 2 of 26 AHF of rats given CD but in none of the 20 AHF fed CS + PHB. These data indicate that CD (which acts as both initiator and promoter) may be responsible for the appearance of aneuploidy. A general model, based on these results and the clonality of each individual focus, is proposed for the development of cells through the preneoplastic stage.

Quantification of collagen in renal glomeruli isolated from human nondiabetic and diabetic kidneys.

Previous studies of diabetic renovascular complications have measured morphologic changes in relatively few glomerular vessels by electron microscopy. The present study samples 20,000 to 80,000 glomeruli from each of ten nondiabetic and ten diabetic age- and sex-matched subjects. Glomeruli were isolated and fractionated by size with a sieving method. Three samples of glomeruli from each subject were analyzed for size, mass, and hydroxyproline content as an index of basement membrane collagen. Approximately 40 per cent of the glomeruli in each sample were isolated. Glomeruli comprised 94 per cent of the tissue elements present, and 92 per cent of the isolated glomeruli were intact. Diabetic glomeruli are larger than nondiabetic glomeruli (mean diameter +/- S.E.M. = 258 +/- 10 mu versus 196 +/- 6 mu) and heavier (499 +/- 63 ng. versus 232 +/- 16 ng.). Diabetic glomeruli have greater hydroxyproline content than nondiabetic glomeruli when content is expressed per glomerulus (21.9 +/- 3.3 ng. versus 7.1 +/- 0.5 ng.) and when expressed per milligram dry weight of glomeruli (44.0 +/- 2.4 mug. versus 31.6 +/- 1.9 mug.). Glomeruli from diabetics of longest duration show the greatest increases in mass and hydroxyproline values. A pathologist's semiquantitative estimation of diffuse glomerulosclerosis revealed a high correlation between hydroxyproline values and histologic determination of the extent of the renal lesion. These measurements allow quantification of basement membrane collagen and may be used to follow development of diabetic vascular complications.

Genomic instability in radial growth phase melanoma cell lines after ultraviolet irradiation.

BACKGROUND/AIMS: Although ultraviolet (UV) irradiation, apoptosis, and genomic instability are all potentially involved in the pathogenesis of melanoma, in vitro studies investigating these changes in the radial growth phase of this neoplasm are still lacking; therefore, this study was designed to investigate these changes. METHOD: An in vitro system consisting of three radial growth phase Wistar melanoma cell lines (WM35, WM3211, and WM1650) was established. Cells were UV irradiated (10 mJ/cm2 for UVB and 6 J/cm2 for UVA), harvested after UV exposure, and evaluated for viability and apoptosis using Trypan blue and terminal deoxynucleotidyl transferase mediated dUTP digoxigenin nick end labelling assays, respectively. Polymerase chain reaction based microsatellite assays were used to examine the cell lines for the presence of microsatellite instability (MSI) using 21 markers at the 1p, 2p, 3p, 4q, 9p, and 17p regions. RESULTS: Exposure to UV initiated progressive cell death associated with pronounced apoptosis, with UVA having a greater effect than UVB. MSI was found in UVB (WM35 and WM3211) and UVA (WM35) irradiated cell lines at 1p, 9p, and 17p, but not in non-irradiated cells. The prevalence of MSI was higher after UVB irradiation (14%) than UVA irradiation (4.7%), and was most frequently found at D1S233. CONCLUSIONS: The ability of erythemogenic UV irradiation to induce both apoptosis and MSI in radial growth phase melanoma cells is suggestive of its role in melanoma pathogenesis. This instability may reflect a hypermutability state, oxidative stress induced DNA damage, replication infidelity, or a combination of these factors.

Aneuploidy and progression in promoted preneoplastic foci during chemical hepatocarcinogenesis in the rat.

Since a number of rat hepatocarcinomas are aneuploid, the model DNA content of enzyme-altered foci was determined cytospectrophotometrically, to assess if ploidy changes occur before cancer is established. Male F344 rats treated with diethylnitrosamine and promoted with a choline-deficient, phenobarbital supplemented diet showed in most enzyme-altered foci a multimodal ploidy distribution with diploid, tetraploid and octoploid peaks. A minority of foci, however, exhibited an aneuploid pattern. This change in ploidy reflects irreversible genomic alterations, indicative of tumor progression. Thus, promotion and progression may coexist simultaneously in this model of carcinogenesis, long before hepatomas can be diagnosed.

Effect of acute administration of N-nitrosopyrrolidine to male Syrian golden hamsters.

A protocol has been developed which decreases the time for administration of N-nitrosopyrrolidine (NPYR) to male Syrian golden hamsters from 25 weeks to a single i.p. injection. Animals were divided into five groups: group I received two 0.5-mmol doses on alternate days; group II was given three 0.33-mmol doses on alternate days; group III received a single dose of 0.5 mmol; group IV was given a single dose of 0.25 mmol and group V served as a control and received saline. Preneoplastic and neoplastic changes in the upper respiratory tract and liver were observed in all carcinogen-treated groups. The number of animals with laryngeal and tracheal tumors in the NPYR-treated groups was dose-dependent. Groups I and II, respectively, had 21 of 26 (81%) and 18 of 24 (75%) animals with either laryngeal or tracheal tumors. Groups III and IV showed 4 of 12 (33%) and 3 of 13 (23%) hamsters with these tumors. No laryngeal or tracheal tumors were observed in control animals. These results indicate that a single dose of NPYR is sufficient to induce respiratory tract tumors in Syrian golden hamsters.

Hepatic veno-occlusive disease after high-dose mitomycin C and autologous bone marrow transplantation therapy.

Three cases of veno-occlusive disease of the liver were diagnosed in four autopsied patients who had received high-dose mitomycin C therapy followed by autologous bone marrow transplantation, and the pathologic finding are reported. Review of 27 liver, examined post mortem, of patients receiving other high-dose chemotherapeutic regimens, 15 of them with subsequent autologous bone marrow transplantation, revealed no evidence of veno-occlusive disease. Veno-occlusive disease may now become a dose-limiting factor in the use of the combined high-dose mitomycin C-bone marrow transplantation therapy. Attention is also drawn to the increasing number of veno-occlusive disease cases being reported in associated with alkylating agents.

Monoclonal antibodies to prothrombin.

Hybridoma technology was used for the production of murine monoclonal antibodies to bovine normal prothrombin. Hybrid cell cultures were assayed for the production of antibodies, both in the absence and presence of calcium ions, by Enzyme-Linked Immunosorbent Assay (ELISA). Antibody-producing cell lines were cloned two times and grown as ascites tumors. Monoclonal antibodies (McAb), isolated by affinity chromatography (Protein A-Sepharose), were tested for their affinity for normal (10-Gla) and dicoumarol-induced abnormal prothrombins containing 2, 5, 7, 8 and 9 gamma-carboxyglutamyl (Gla) residues. A total of 24 McAb were obtained and the immunoglobulins were of the IgG1 subclass. Nine of the twenty-four McAb did not require Ca2+ for the formation of Ag-Ab complexes, and reacted equally with normal and Gla-deficient prothrombins. These antibodies had affinity for prethrombin1 (P1) but not for the Gla-containing prothrombin fragment1 (F1) portion of the molecule. In contrast, the 15 Ca2+-dependent McAb reacted with F1 but not with P1. They discriminated the abnormal prothrombins based upon their Gla content. For example, though all the Ca2+-dependent McAb formed Ag-Ab complexes with 9-, essentially none formed with 5- or less-Gla prothrombins. [Some reacted equally with 9- and 10-Gla (normal) prothrombin while others had only 25% of normal affinity for 9-Gla isomer]. Only four and twelve of the 15 McAb had some affinity for 7- and 8-Gla variants, respectively. These results show that antibodies which react with the Ca2+-stabilized conformation of prothrombin are not specific for normal prothrombin, as has been reported in the literature.

Characterization of chloramphenicol- and 8-azaguanine-resistant mutants isolated from a continuous rat-liver epithelial cell line.

2 non-tumorigenic, chloramphenicol- and 8-azaguanine-resistant strains have been isolated from the rat-liver cell line K-22, by a 2-step mutagenesis procedure. Their chromosome composition and growth properties have been characterized. Failure of chloramphenicol to inhibit mitochondrial protein synthesis in one of the clones, F1, strongly suggests that resistance to the antibiotic in this strain is due to a mutation in mitochondrial DNA.

Prestaining of membrane markers to identify specific areas for Feulgen cytophotometric determinations in a single section.

In cytospectrophotometric determinations of the nuclear DNA content in tissues, two consecutive sections are commonly employed: one stoichiometrically stained (as with the Feulgen reaction) for the actual measurements and a second routinely stained (as with hematoxylin and eosin) to define the limits of abnormal areas. This paper proposes the use of stainable cell membrane markers to identify the boundaries of such areas in only the one section in which DNA measurements are to be performed. The use of this procedure for the analysis of enzyme-altered foci and preneoplastic nodules in the rat liver is described. The membrane marker staining, which does not affect the nucleus or cytoplasm, does not interfere with the nuclear DNA determinations.

Is 8-hydroxydeoxyguanosine a mediator of carcinogenesis by a choline-devoid diet in the rat liver?

The mechanism(s) by which a diet devoid of choline (CD) induces hepatocellular carcinomas in rats remains unknown. Although animals fed this diet develop nuclear lipid peroxidation, suggesting oxidative DNA damage, there is no direct evidence that this occurs. In this study, 8-hydroxydeoxyguanosine (8-OHdG), a DNA adduct generated by reactive oxygen species, was analyzed in the liver of rats fed a CD diet and in controls receiving a choline-sufficient (CS) diet. After partial hepatectomy, the animals were injected with diethylnitrosamine (DEN, 50 mg/kg body wt) or with saline and fed a CD or CS diet for 24 weeks. While liver DNA from rats injected either with DEN or saline and fed a CS diet did not show detectable amounts of the nucleotide, those who were fed DEN/CD and saline/CD demonstrated similar, easily measurable levels of 8-OHdG. These results indicate that there is a positive association between the continuous administration of a CD diet and the production of 8-OHdG in liver DNA, and support the idea that oxidative DNA damage is involved in carcinogenesis by a CD diet.

Intermediate filaments of Schwann cells.

Intermediate filaments were prepared from distal stumps of rabbit sciatic nerve 5 weeks after nerve section, at which time Schwann cells account for 85--90% of the cell area. A polypeptide of molecular weight 58,000 was the main component of this fraction. An antiserum raised in guinea pig against this polypeptide stained all cells present in the distal stump, as well as Schwann cells and 3T3 cells in culture. The identity of the molecular weight 58,000 polypeptide obtained from distal stumps with vimentin was proved with one and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and with immunoautoradiography. It is concluded that the intermediate filament subunit of undifferentiated Schwann cells is vimentin. The possibility that Schwann cells in normal nerve may have another type of intermediate filament besides vimentin cannot be ruled out.

The alloantibody response in the allogeneically pregnant rat. IV. Analysis of the alloantibody specificities with monoclonal antibodies.

We have compared the serum alloantibody population from female rats immunized either by allogeneic pregnancies or by conventional immunizations. The only allogeneic difference in both types of immunization was class I of the MHC. Pregnancy-induced alloantibodies as compared with conventionally raised alloantibodies were more homogeneous with respect to isoelectric point, and were more homogeneous as defined by competition experiments with anti-class I monoclonal antibodies. The genetic control of the pregnancy-induced alloantibody response was also verified.

Differential responses of two inbred rat strains to a choline-deficient diet during liver carcinogenesis.

We examined the effect of a choline-devoid (CD) diet on the development of gamma-glutamyltranspeptidase (GGT)-positive foci in both sexes of the inbred rat strains Fischer 344 and PVG/R8. Following partial hepatectomy, 7 to 8 week old animals were given a choline-supplemented diet for 1 week. Two groups were then formed: one remained on choline-supplemented diet as control, and the other was switched to the CD diet. The animals were killed 10, 16 and 24 weeks later. Liver samples were then stained with hematoxylin-eosin and Masson's trichromic, and histochemically analyzed for GGT. Fatty degeneration and collagen formation was severe in F344 males while it was mild or absent in F344 females and in both sexes of PVG rats. Stereochemical quantitation showed that F344 males had a significantly greater increase in the number of positive liver foci (as well as in their mean volume and the percentage of liver occupied by them) than F344 females and PVG animals of either sex (P < 0.01). These results suggest that not only sex but also the genotype of the host plays a role in the different responses to a CD diet. In depth analysis of these factors should prove valuable to investigate this dietary model of hepatocarcinogenesis further.

In vitro cytotoxicity and in vivo acute responses to some amino acid copolymers and their esters.

The present study was performed in order to establish if there is any correspondence between specific parameters of tissue reaction to implanted biomedical materials and in vitro cytotoxicity. The presence of various types of inflammatory cells and/or necrosis in rats implanted subcutaneously with a series of 42 alpha-amino acid copolymers and their esters was compared with their in vitro toxic effects, as determined by an agar overlay technique. Only necrosis appeared to correlate with reactions scored as strongly positive by tissue culture procedures. Future studies with slightly toxic materials should assess if tissue culture methods could be helpful in predicting other levels of reaction at the polymer-tissue interface.

Alterations of mismatch repair protein expression in benign melanocytic nevi, melanocytic dysplastic nevi, and cutaneous malignant melanomas.

Immunoperoxidase-staining methods were used to examine the expression of hMLH1, hMSH2, and hMSH6 mismatch repair (MMR) proteins in 50 melanocytic lesions. Microsatellite instability (MSI), screened previously in these lesions by polymerase chain reaction-based microsatellite assay, showed low-level microsatellite instability (MSI-L) in 11 of 22 melanocytic dysplastic nevi (MDN) and two of nine primary cutaneous malignant melanomas (CMMs) but not in the benign melanocytic nevi (BN). Mismatch repair proteins were widely expressed in the epidermis and adnexal structures. All lesions showed positive immunoreactivity with a gradual decrease in the MMR staining values during the progression from BN to MDN to CMMs. The average percentage of positively (PP) stained cells for hMLH1, hMSH2, and hMSH6 in BN was 85.50 +/- 1.95, 77.90 +/- 4.50, and 87.11 +/- 1.85, respectively. The PP cell values in CMMs were significantly reduced as compared with BN (75.22 +/- 3.57, p= 0.01; 56.11 +/- 8.73, p= 0.02; 65.22 +/- 6.47, p = 0.0002 for hMLH1, hMSH2, and hMSH6, respectively). No comparable significant difference was found between microsatellite stable and MSI-L lesions (p = 0.173, p = 0.458, and p = 0.385), suggesting a lack of correlation between MMR expression and MMR function. There was a direct correlation between PP cell values of hMSH2 and hMSH6 (R = 0.39, p = 0.008), implying that their expression could be regulated by a common mechanism. Thus, an important finding of these studies was the reduction of MMR protein levels in CMMs; whether this reflects underlying genetic or epigenetic mechanisms is still to be determined.

Comprehensive analysis of 112 melanocytic skin lesions demonstrates microsatellite instability in melanomas and dysplastic nevi, but not in benign nevi.

INTRODUCTION: the length of DNA repetitive sequences (microsatellite instability (MSI)) represent distinct tumorigenic pathways associated with several familial and sporadic tumors. MATERIAL AND METHODS: To investigate the prevalence and frequency of MSI in melanocytic lesions, the polymerase chain reaction (PCR)-based microsatellite assay was used to examine formalin-fixed, paraffin-embedded tissues of 30 benign melanocytic nevi, 60 melanocytic dysplastic nevi (MDN), and 22 primary vertical growth phase cutaneous malignant melanomas (CMM). Twenty-four microsatellite markers at the 1p, 2p, 3p, 4q and 9p chromosomal regions were used. RESULTS: MSI was found at 1p and 9p in MDN and CMM but not in benign melanocytic nevi. The overall prevalence of MSI was 17/60 (28%) in MDN and 7/22 (31%) in CMM. The frequency of MSI ranged from 2/24 (9%) to 4/24 (17%) and was most commonly found at D9S162. There was a statistically significant correlation between degree of atypia and frequency of MSI (p<0.001) in MDN. There were two MSI banding patterns: band shifts and additional bands. CONCLUSIONS: The data presented revealed the presence of low-frequency MSI (MSI-L) at the 1p and 9p regions in both MDN and CMM. Whether the MSI-L pattern reflects a defect in mismatch repair genes is still to be determined.