Immunohistochemical demonstration of a mRNA-transport protein in rat liver putative preneoplastic foci.

A 65K protein, known for promoting nucleocytoplasmic mRNA transport in a cell-free system, was previously found in fetal and tumor cells of the rat. The primary objective of this study was to show specificity of immunohistochemical staining for the 65K protein in the livers of rats subjected to a hepatocarcinogenesis protocol. Altered hepatic foci were induced by feeding male weanling Sprague-Dawley rats 2-acetylaminofluorene (AAF) followed by a phenobarbital (PB) diet. It was shown, using polyclonal antibodies produced in rabbits, that the 65K protein was present in the cells of rat liver putative preneoplastic foci, with little or none being detected in the surrounding cells.

Repression by sustained-release beta-glucuronidase inhibitors of chemical carcinogen-mediated induction of a marker oncofetal protein in rodents.

The degree of induction of an oncofetal protein marker in rodents by selected chemical carcinogens has been correlated with changes in carcinogenicity induced by dietary D-glucaro-1,4-lactone (GL) based anticarcinogens. These potent anticarcinogens may act to increase the clearance of carcinogens as glucuronides through the inhibition of beta-glucuronidase. The sustained-release forms are particularly effective, 1.5 mmol/kg of GL maintaining serum beta-glucuronidase activity at or below 50% for only 1 h, while an equivalent amount of calcium glucarate (CGT) maintained this level of inhibition for over 5 h. CGT or other sustained-release inhibitors, when fed to rodents during administration of carcinogens that undergo glucuronidation, caused a marked reduction in the induction of the marker protein. For those systems where other markers of carcinogenesis were also assessed, it was determined that the inhibition of marker-protein induction was quantitatively similar to both the inhibition of binding of the carcinogen to DNA and the subsequent induction of tumors in target organs.

Effects of calcium glucarate on the promotion of diethylnitrosamine-initiated altered hepatic foci in rats.

Calcium glucarate (CGT), an inhibitor of beta-glucuronidase, is a potent inhibitor of chemically-induced tumors when administered orally. The present study was undertaken to determine the effects of CGT on the promotion of hepatocarcinogenesis by phenobarbital following initiation with diethylnitrosamine (DENA). Partially hepatectomized, DENA-initiated female Sprague-Dawley rats, previously maintained only on chow diet for 2 months, were supplemented with either 0.05% phenobarbital alone or 0.05% phenobarbital plus 4% dietary CGT, for varying time intervals up to 6 months. Histopathologic evaluation of the liver sections showed that CGT significantly delayed the development of altered hepatic foci (AHF). By the seventh month post-initiation, however, the frequency and severity of changes seen in the livers of experimental animals approximated those of the controls.

Correspondence between biochemical and antigenic activity of a 60 kilodalton oncofetal protein during carcinogenesis and tumorigenesis.

Biochemical and immunochemical (ELISA) assays were used concurrently to monitor the rat plasma concentration of a recently identified 60 kDa oncofetal protein. Both the biochemical and antigenic activities increased and decreased in parallel during the growth and after surgical removal, respectively, of a solitary transplantable tumor. These activities also responded similarly in response to the specific induction of the 60 kDa factor in the rat by chemical carcinogens. The data indicate that both assays give similar results and provide further evidence for the specificity of the 60 kDa factor as a marker for carcinogenesis and tumorigenesis.

Dietary glucarate-mediated reduction of sensitivity of murine strains to chemical carcinogenesis.

Serum beta-glucuronidase activity is shown to differ quantitatively in the following strains of mice, listed in order of increasing activity: C3H, C57BL/6 less than BALB/c, DBA/2, ICR less than SENCAR, A/He. The level of the enzyme in the murine strains is shown to correlate with the urinary excretion of 17-ketosteroids, which in turn reflects the endogenous level of androgens. Dietary calcium D-glucarate, an in vivo beta-glucuronidase inhibitor, reduced the steady state level of both beta-glucuronidase and 17-ketosteroid excretion in the highly susceptible A/He and SENCAR strains to that of strains known to be resistant to chemical carcinogenesis. Sensitivity of the A/He strain is significantly reduced by dietary calcium glucarate, which is shown to inhibit DNA binding and the induction of pulmonary adenomas by benzo[a]pyrene.

Dietary glucarate as anti-promoter of 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis.

Using as a criterion the inhibition of serum beta-glucuronidase activity, dietary calcium D-glucarate is shown to serve as an efficient slow-release source in vivo of D-glucaro-1,4-lactone, the potent endogenous inhibitor of this enzyme. Using the 7,12-dimethylbenz[a]anthracene model of mammary tumor induction in rats it is shown for the first time that feeding the rats calcium D-glucarate-supplemented diet after treatment with the carcinogen, inhibits tumor development by over 70%. Supportive evidence is presented for the theory that calcium D-glucarate inhibits or delays the promotion phase of mammary carcinogenesis by lowering endogenous levels of estradiol and precursors of 17-ketosteroids. Therefore, dietary glucarate can be used to lower blood and tissue levels of beta-glucuronidase, and in turn of those carcinogens and promoting agents which are excreted, at least in part, as glucuronide conjugates.

A 60 kilodalton oncofetal protein as tumor marker.

A 60 kd oncofetal protein, shown previously to be present in tumor cytosol and rat embryonic tissue, has been measured by a specific assay in the blood plasma of 42 cancer patients with tumors at 12 different sites. All 42 cancer patient plasmas were positive, while the plasmas from 20 normal controls were negative. Similar results were obtained with the experimental animal system. The protein is present in low concentrations in cancer patient plasma, amounting to less than 0.008% of the total plasma protein. Following complete surgical removal of the tumor, the plasma concentration of the factor drops rapidly. In the rate the biological half-life was 8 days.

beta-Glucuronidase levels in patients with fibrocystic breast disease.

Certain enzymes in tissues and body fluids may, through reversal of the detoxification process, influence the composition and availability of steroid hormones, toxins, and carcinogens. The ubiquitous enzyme beta-glucuronidase, which hydrolyzes glucuronide conjugates, thereby reversing one of the main detoxification and excretion pathways, was found to vary in concentration in different cysts over a 300-fold range. The distribution was a continuum, devoid of discrete sub-populations. Evidence obtained on selected cyst fluids of high and low beta-glucuronidase activities indicated that the level of the enzyme significantly influenced the ratio of unconjugated: glucuronidated estradiol. The patients with fibrocystic breast disease fell into 2 distinct subpopulations on the basis of their serum beta-glucuronidase activity. In one group the activity was near normal, while in the second group the average serum beta-glucuronidase activity was 3-fold higher than in the women who did not have benign breast disease.

Chemical carcinogens as specific inducers of a 60-kilodalton oncofetal protein in rats.

A 60-kd oncofetal protein which is released to circulation in vivo from tumor cells, or from normal tissues following carcinogen-treatment, has been evaluated against single dose regimens of 18 known chemical carcinogens and 13 known chemical non-carcinogens. The carcinogens included genotoxic and presumed non-genotoxic compounds and the blood plasma 21 days post-treatment of rats with the chemicals was measured. All carcinogens tested transiently induced the 60-kd factor, while none of the non-carcinogens, which included non-carcinogenic analogs or toxins, induced the factor. The results suggest that the early induction of this oncofetal protein may be an indicator of carcinogenicity. The induction of reversible phenotypic changes in normal cells by tumor promoters did not induce the 60-kd factor. Following the carcinogen-mediated transient induction, persistent production of the 60-kd factor occurred at later times if a tumor developed.

Immunological identity of a 60 kd oncofetal protein induced in rats by chemical carcinogens and released by transformed cells.

A 60,000 dalton (60 kd) oncofetal protein was previously shown to be produced by tumors in tumor-bearing rats and by target tissues within 3 weeks of carcinogen treatment. The factor is released to and accumulates in the blood in vivo and in the conditioned medium of cultured transformed cells in vitro. A polyclonal antibody produced against the 60 kd factor purified from the plasma of a rat carrying the N-2-fluorenylphthalamic acid-induced transplantable Hepatoma 7777, was tested against the 60 kd factor from various sources. Based on the results of immunoprecipitation of biochemical activity associated with the 60 kd factor, it was determined that these anti-60 kd antibodies cross-reacted with the factor released by a dimethylbenzanthracene-induced rat mammary carcinoma, with the factor in rat tumor cytosol and with rat spontaneous lymphoma cells, but not with a 60 kd factor isolated from pooled cancer patient plasma. Furthermore, these antibodies cross-reacted with the 60 kd factor induced within 21 days of treatment of the rats with a range of carcinogens from 8 chemical structural groups. The anti-60 kd factor antibodies did not cross-react with a 35 kd factor having similar biochemical activity found in normal adult cells.

Inhibition of 7,12-dimethylbenzanthracene-induced rat mammary tumorigenesis by 2,5-di-O-acetyl-D-glucaro-1,4:6,3-dilactone, an in vivo beta-glucuronidase inhibitor.

2,5-Di-O-acetyl-D-glucaro-1,4:6,3-dilactone ( DAGDL ) is a slow release form of D-glucaro-1,4-lactone (GL), a non-toxic natural inhibitor of beta-glucuronidase. When administered orally to female rats in conjunction with a carcinogenic dose of 7,12-dimethylbenzanthracene (DMBA), this compound caused a 70% reduction in the number of rats with mammary tumors and 72% reduction in the number of mammary tumors per rat. Co-administration also reduces the induction by DMBA of a 60 kd oncofetal protein, previously shown to be associated with carcinogenesis and tumorigenesis. DAGDL administration depressed beta-glucuronidase activity both in the absence and presence of concurrent treatment with DMBA and also markedly reduced binding of DMBA to organ DNA. The anti-carcinogenic effect of DAGDL appears to be independent of route of administration of DMBA. It is proposed that inhibition of beta-glucuronidase increases the proportion of DMBA which is sequestered and excreted as the glucuronide and therefore unavailable for activation to the proximal carcinogen.

Endogenous and exogenous factors affecting ribosomal RNA release from rat liver nuclei in a cell-free system.

rRNA release from isolated liver nuclei has been analyzed in a reconstituted cell-free system using density-gradient analysis and hybridization to a specific recombinant DNA probe to monitor the process. The cell-free system was shown previously to be energy- and cytosol-dependent and to support the formation and release of functional ribosomal subunits. The release of rRNA is now shown to have an absolute dependence on a 70000 dalton cytosol protein. Although in vivo studies suggest that chronic administration of thioacetamide may block formation of a protein involved in the nucleocytoplasmic transfer of ribosomes, the 70000 dalton transport factor is not affected by the treatment. Rather the defect appears to be localized to the nucleus, since it cannot be reversed with normal cytosol from a homologous source. Early stages of nRNA processing appear to be affected by thioacetamide, although additional effects on transport are not ruled out.

Nucleocytoplasmic release of repetitive DNA transcripts in carcinogenesis correlates with a 60 kilodalton cytoplasmic protein.

Treatment of rats with the hepatocarcinogen 3'-methyl-4-dimethylamino-azobenzene (3'-MeDAB) causes the appearance in the liver cytosol of a 60 kilodalton oncofetal protein. The appearance of this factor occurs within 40 h of treatment and coincides with the increase in the amount of rapidly labeled RNA released from nuclei in a reconstituted cell-free system. Cross-over experiments show that this increase is due to an enhanced transport capacity of the cytosol. The 60 kilodalton RNA transport factor is also present in the cytosol of tumor cells. Addition of the 60 kilodalton factor to normal liver cytosol causes the transport of repetitive RNA sequences similar to those transported from liver nuclei to tumor cell cytosol and those transported to the tumor cell cytoplasm in vivo. This factor modifies nuclear RNA restriction, at least in part, by eliciting the transport of repetitive RNA normally retained within the nucleus of the normal cell.

Characterization of a 60,000-dalton oncofetal protein from the plasma of tumor-bearing rats.

A tumor cell-associated protein, previously shown to be present in the circulation of carcinogen-treated and tumor-bearing animals and cancer patients, has now been identified in the cytosol of embryonic tissue. This oncofetal protein, which is absent from the plasma of normal animals, has been purified from the plasma of tumor-bearing rats by a series of steps including ammonium sulfate fractionation and chromatography on Sepharose CL-6B and on CM Affi-Gel Blue. The tumor and fetal-associated 60-kd rat factors appear to be identical based on their reactivity to polyclonal antibody produced against the tumor factor. The factor, assayed by its ability to induce the transport of RNA from isolated nuclei, is a phosphoprotein with a minimum molecular weight of 60,000, as determined by polyacrylamide gel electrophoresis. In its purified form it is phosphorylated in the presence of the catalytic subunit of heart muscle protein kinase and ATP but does not exhibit auto-phosphorylating activity. 32P-orthophosphate is also incorporated into the phosphoprotein in vivo.

Absence of the cancer-associated factor with a molecular weight of 60,000 from the plasma of patients with a spectrum of nonneoplastic conditions.

We demonstrated previously (Cancer Res., 42: 4964-4969, 1982) that a tumor-associated factor was consistently present in the plasma of over 100 human cancer patients with tumors at 31 different sites. The plasma of healthy controls had very low activity in the biochemical assay. In the present study, we show by a combination of molecular sieving and assay of nuclear RNA transport that the tumor-associated factor, which has a molecular weight of 60,000, is undetectable in the plasma of healthy adults. The low activity reported earlier is due to three normal cell factors of markedly different molecular weight. Furthermore, the tumor factor is shown to be absent from the plasma of male and female patients hospitalized for a variety of nonmalignant surgical conditions. Only the plasma from patients who were pregnant, suffered from chronic renal failure, or had recent myocardial infarction gave false positives in the biochemical assay. However, in these cases, the activity was due to an increase in the normal tissue-associated factors and not to the appearance of the Mr 60,000 tumor-associated factor. The factor is present in amniotic fluid, confirming that it is a fetal factor which does not cross the placental barrier. Thus, it may be classified as an oncofetal factor. All four factors found in the plasma were identified in the cytosol from a human tumor. In summary, the tumor-associated factor appears to be tumor specific and can be unambiguously identified by bioassay of the plasma factors eluting from Sepharose CL-6 B columns in the Mr 60,000 region. It can also be identified by examination of sodium dodecyl sulfate:polyacrylamide gel electrophoretograms of the appropriate Sepharose CL-6 B fractions after removal of albumin.

An oncofetal 60-kilodalton protein in the plasma of tumor-bearing and carcinogen-treated rats.

A Mr 60,000 protein, detected by its ability to induce the release of RNA from isolated nuclei, is present in the plasma of tumor-bearing and carcinogen-treated rats, together with low amounts of 2 messenger RNA transport proteins identified earlier in normal cells. The Mr 60,000 protein has been identified in tumor cell cytoplasm and in amniotic fluid, but does not appear to cross the placental barrier. Significant amounts of the Mr 60,000 oncofetal protein appear in the plasma of carcinogen-treated rats within a few weeks of treatment. It may be the fetal form of the adult messenger RNA transport proteins.

Use of adenosine 5'-0-(2-thiotriphosphate) for revealing of newly initiated transcripts in isolated rat liver nuclei.

Initiation of DNA-dependent RNA synthesis in isolated rat liver nuclei was studied with adenosine 5'-0-(2-thiotriphosphate), (beta-S-ATP), as a precursor. The newly made RNA labelled with sulfur at 5'-triphosphate termini (thio-RNA) was isolated by affinity chromatography on a mercury-agarose column. Sulfur label can be removed from thio-RNA by digestion with phosphodiesterase I and nucleotide pyrophosphatase. Gel electrophoresis revealed that thio-RNA synthesized during 30 min was composed of 4S-35S molecules with three prevailing classes grouped around 4S-5S, 16S and approximately 35S. Differential sensitivity of the thio-RNA classes to low (1 microgram/ml) and high (200 micrograms/ml) concentrations of alpha-amanitin disclosed that beta-S-ATP was used for initiation of transcription by all three classes of RNA polymerases, and that thio-RNA included molecules as large as 18S initiated by RNA polymerase II. Thio-RNA resistant even to high doses of alpha-amanitin represents probably a product of RNA polymerase I which was initiated and elongated up to 35S.

Separation of mercury substituted RNA synthesized in isolated rat liver nuclei.

The population of RNA molecules synthesized in isolated rat liver nuclei in vitro in the presence of [3H]CTP and Hg-UTP was successfully fractionated into at least two subfractions containing various proportions of mercury label. Fractionation was achieved either by step-wise chromatography of Hg-RNA on thiopropyl-Sepharose columns or by density gradient centrifugation in metrizamide. The fraction of RNA heavily labeled with Hg-UTP was composed mainly of 4--18S RNA and contained virtually all radioactivity derived from [gamma-32P]ATP or [gamma-32P]GTP. The slightly mercurated RNA fraction consisted mainly of longer RNA molecules (12- greater than 28S) and was not labeled with [gamma-32P]ATP or [gamma-32P]GTP. Labeling with gamma-32P nucleoside triphosphates was sensitive both to rifamycin AF/013 and heparin whereas labeling with [3H]CTP was fully resistant to the inhibitors and showed sensitivity to low doses of alpha-amanitin. We assume that the observed subpopulation of heavily mercurated RNAs consists of RNA molecules initiated in vitro.