The role of skin painting in predicting lung cancer.

The mouse skin cancer model provides an important system for studying mechanisms involved in the various stages of carcinogenesis and for bioassaying tobacco smoke constituents and additives for carcinogenic/cocarcinogenic and tumor-promoting properties as well as for identifying compounds that may inhibit tumor formation and malignant conversion. In addition, it is an excellent model for studying the formation of precancerous lesions as well as squamous cell carcinomas. It relates very well to other squamous cell carcinoma models and contributes to better understanding of the human epithelial cancers including lung cancer. The SENCAR mouse is an established model system demonstrated to be more sensitive than the B6C3F1 or Swiss CD-1 strains in the initiation/promotion skin-painting test method. Although the relationship between mouse skin tumors and any manifestation of the toxicity of tobacco smoke and other complex environmental mixtures in humans is unknown, the skin-painting model is the only assay that provides a practical method of obtaining a tumorigenic end point with cigarette smoke condensates and other complex mixtures. This assay provides a rapid response with relative ease of quantification of various parameters of tumorigenic response including tumor incidence, latency, multiplicity, and malignancy.

Avicins, a family of triterpenoid saponins from Acacia victoriae (Bentham), suppress H-ras mutations and aneuploidy in a murine skin carcinogenesis model.

We tested the ability of avicins, a family of triterpenoid saponins obtained from Acacia victoriae (Bentham) (Leguminosae: Mimosoideae), to inhibit chemically induced mouse skin carcinogenesis. Varying doses of avicins were applied to shaved dorsal skin of SENCAR mice 15 min before application of 100 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) twice a week for 4 weeks (complete carcinogenesis model). The dorsal skin of a second group of mice was treated with one dose of 10 nmol of DMBA. Avicins were then applied 15 min before repetitive doses of 2 microg of phorbol 12-tetradecanoate 13-acetate (TPA) twice a week for 8 weeks (initiation/promotion model). At 12 weeks, avicins produced a 70% decrease in the number of mice with papillomas and a greater than 90% reduction in the number of papillomas per mouse in both protocols. We also observed a 62% and 74% reduction by avicins in H-ras mutations at codon 61 in the DMBA and DMBA/TPA models, respectively, as well as a significant inhibition of the modified DNA base formation (8-OH-dG) in both protocols. Marked suppression of aneuploidy occurred with treatment at 16 weeks in the initiation/promotion experiment. These findings, when combined with the proapoptotic property of these compounds and their ability to inhibit hydrogen peroxide (H(2)O(2)) generation, nuclear factor-kappaB (NF-kappaB) activation, and inducible nitric oxide synthase (iNOS) induction reported elsewhere, suggest that avicins could prove exciting in reducing oxidative and nitrosative stress and thereby suppressing the development of human skin cancer and other epithelial malignancies.

Inhibition of azoxymethane-induced rat colon carcinogenesis by potassium hydrogen D-glucarate.

While calcium D-glucarate was shown to inhibit chemical carcinogenesis in various animal models, the effect of potassium hydrogen D-glucarate has not been extensively investigated. In the present study, potassium hydrogen D-glucarate markedly inhibited azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. Potassium hydrogen D-glucarate (PHG) or potassium hydrogen carbonate (PHC) were administered to rats in a diet (140 mmol/kg). Continual post-initiation treatment with potassium hydrogen D-glucarate reduced both tumor incidence and multiplicity at sacrifice by ca. 60%, while PHC had no effect. amelioration of overexpression of the betaG gene in rat colon carcinomas was observed using RT-PCR and Northern blot analysis. We hypothesize that previously demonstrated conversion of PHG to D-glucaro-1,4-lactone, a potent inhibitor of beta-glucuronidase (betaG), may be responsible for this effect. The mechanism of PHG inhibition of colon carcinogenesis may also involve suppression of cell proliferation and possibly alterations in cholesterol synthesis or cholesterol metabolism to bile acids. In conclusion, PHG possesses excellent potential as a natural, apparently non-toxic inhibitor to prevent colon cancer.

Metabolism, uptake, and excretion of a D-glucaric acid salt and its potential use in cancer prevention.

D-Glucaric acid (GA) is a nontoxic, natural compound. One of its derivatives is the potent beta-glucuronidase inhibitor D-glucaro-1,4-lactone (1,4-GL). The goal of this study was to demonstrate the in vivo formation of 1,4-GL from a D-glucarate salt and determine its metabolism, uptake by selected organs, and excretion following oral administration of potassium hydrogen D-[14C]glucarate to male and female Sprague-Dawley rats. 1,4-GL increases detoxification of carcinogens and tumor promoters/progressors by inhibiting beta-glucuronidase and preventing hydrolysis of their glucuronides. 1,4-GL and its precursors, such as potassium hydrogen D-glucarate and calcium D-glucarate, may exert their anticancer action, in part, through alterations in steroidogenesis accompanied by changes in the hormonal environment and the proliferative status of the target organ. Thus, GA derivatives may be useful as new or adjuvant cancer preventive and therapeutic agents. In our study, 1,4-GL was found to be formed from the D-glucarate salt in the stomach of rats. It was apparently absorbed from the gastrointestinal tract, transported with the blood to different internal organs, and excreted in the urine and to a lesser extent in bile. There were no significant differences in the metabolism of PHG between male and female rats. Thus, formation of 1,4-GL from D-glucaric acid derivatives may be prerequisite for their inhibition of chemical carcinogenesis in rodents and prevention of breast, prostate, and colon cancer in humans.

Expression of an oncofetal 65-kDa phosphoprotein in lymphocytic and granulocytic leukemias.

We examined the expression of an oncofetal 65-kDa phosphoprotein, termed p65, in patients with lymphocytic and granulocytic leukemia. This protein was previously identified in rat fetal tissues and in epithelial cancers of rat and human origin. Using the anti-p65 monoclonal antibodies MB2 and MF11 in a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), we analyzed the expression of the protein in sera of 80 normal, healthy controls and in 61 patients with benign, nonneoplastic diseases. We established that the upper level of normal p65 concentration is 115 U/ml p65 (mean plus two standard deviations above the mean in a control group). We also analyzed p65 levels in sera of 71 patients with leukemia in different stages of development. The level of p65 was well above normal in 95% of acute lymphocytic leukemia (ALL; 19 cases), 83% of acute myeloblastic leukemia (AML; 23 cases), 37% of chronic lymphocytic leukemia (CLL; 19 cases), and 30% of chronic myelogenous leukemia (CML; 10 cases). MB2 monoclonal antibodies were used for immunocytochemical staining of isolated lymphocytes from normal peripheral blood and from blood of leukemic patients (in 12 CLL patients, the p65 positivity was 83%, in 2 ALL patients, 100%, and in 4 AML patients, 75%). Our data suggest that p65 protein may be of use as a tumor marker in leukemia.

Presence in bovine fetal serum of the protein antigenically related to p65-tumor associated antigen: its isolation and polyclonal antibody production.

Monoclonal antibodies raised to the 65-kDa tumor-associated protein (p65) isolated from a human breast cancer cell line have been used to detect an antigenically related protein (p65-like) present in fetal bovine serum (FBS) by Western blot analysis. We have isolated the p65-like protein from FBS by isoelectrofocusing (IEF) on native gels followed by electrophoresis in 12.5% polyacrylamide gel containing 0.1% SDS (SDS-PAGE). Immunostaining with anti-p65 monoclonal antibody of fetal bovine serum fractions separated by electrophoresis on cellulose acetate membrane revealed that the p65-like protein had a location similar to one of gamma-globulin. This protein migrates as a single band upon electrophoresis in SDS-PAGE and had four isoforms which migrate as two doublets with pI's of approximately 5.0 and 5.3.

The oncofetal protein p65: a new member of the steroid/thyroid receptor superfamily.

The 65-kDa oncofetal protein (p65), a potential tumor marker discovered and characterized in our laboratory, is highly conserved in different species. Its amino acid composition, peptide map, and N-terminal and internal peptide sequences are very similar if not identical in humans and rodents. We have now identified the p65 gene as a novel member of the superfamily of genes that encode nuclear receptors for various hydrophobic ligands such as steroids, vitamin D, retinoic acid, and thyroid hormones. these receptors are composed of several domains important in hormone binding, DNA binding, dimerization, and transcription activation. The human p65 cDNA was partially cloned, revealing at its C-terminal end regulatory elements typical of this superfamily of genes. The DNA-binding domain coincides with the cysteine-rich region encompassing the two conserved zinc fingers. In addition, the domain homologous to the receptor dimerization site was found close to the C-terminal end. The p65 protein is highly homologous to estrogen receptor in its DNA-binding domain but not in other regions of the sequence, indicating that p65 is a new receptor with an as yet unknown ligand. In addition, we have identified in the cloned p65 cDNA fragment sequences encoding two peptides, obtained by CNBr cleavage, whose amino acid sequences were previously established.

Serological and immunohistochemical detection of a 65-kDa oncofetal protein in breast cancer.

Sera from 132 breast cancer patients and 112 healthy female controls were tested with a double-antibody sandwich ELISA using two different monoclonal antibodies against the 65-kDa oncofetal protein, termed p65. Of cancer sera, 90.2% were positive for p65. The average level of p65 was 466.5 +/- 243.8 ng/ml (mean +/- S.D.) in carcinomas and 37.4 +/- 29.5 ng/ml (mean +/- S.D.) in controls (P < 0.0005). A selected group (n = 15) of these 132 patients were needle-biopsied and assessed immunohistochemically using monoclonal antibodies against p65. Nucleocytoplasmic expression was found in 12 patients (80%) using monoclonal antibodies. Expressions of p65 were concordant in 13 (86%) cases between serum and tumour tissues, but did not correlate with tumour DNA ploidy, histological grade or hormone receptors levels. Sera were also tested for CA 15-3 with the average value in cancer serum being 132.4 +/- 14.0 U/ml; there was no significant concordance between the two markers. Thus, p65 may be a potential serum and/or immunohistochemical marker for breast carcinoma.

Expression of a 65 kda oncofetal phosphoprotein in the altered hepatic foci of rats fed 2-acetylaminofluorene followed by phenobarbital.

The altered hepatic foci (AHF) developed in livers of Sprague-Dawley male rats,by acetylaminofluorene/phenobarbital protocol, were analyzed on paraffin sections for gamma-glutamyl-transpeptidase, glutathione S-transferase P and 65 kDa oncofetal protein (p65). 92% percent of the foci were GST-P-positive, 89% of those were also positive for GGT but only 10-12% were positive for p65. Comparison of tissues from different sources such as fetal and normal liver, foci, nodules, surrounding parenchyma and hepatocellular carcinomas revealed the presence of p65 protein in all tissues except normal rat liver and liver parenchyma surrounding the focal lesions.

Comparative structural analysis of human and rat 65 kDa tumor-associated phosphoproteins.

1. A 65 kDa-tumor-associated protein (p65) was isolated from human and rat carcinoma cell culture media. Antibodies raised to the rat protein recognized an antigenically related protein in human cancer cell line. 2. Amino acid composition, N-terminal and internal sequence as well as peptide map and western blot analysis of the p65 strongly suggest a high degree of homology between the human and rat p65 proteins. 3. Homology searches indicated that p65 was not homologous to previously sequenced proteins, but that it may be related to proteins of the steroid receptor superfamily of genes, especially c-erb A gene.

Demonstration of a 65 kDa tumor-specific phosphoprotein in urine and serum of rats with N-methyl-N-nitrosourea-induced mammary adenocarcinomas.

Polyclonal antibodies against a 65 kDa tumor-associated phosphoprotein (p65) were used to develop an ELISA to analyze the presence of p65 in urine and serum of rats bearing N-methyl-N-nitrosourea-induced mammary gland adenocarcinomas. Highly purified rat p65 was added to normal urine and serum to establish a quantitative standard curve with the average correlation coefficient being 0.98 and 0.99 respectively. All samples of urine and serum obtained from different carcinoma-bearing rats showed p65 concentrations above the normal levels found in the control urine and sera. The correlation coefficient between tumor burden and p65 concentration in urine and serum was 0.65 and 0.77 respectively. The average levels of p65 in normal urine and normal serum were 37.0 +/- 32.0 and 48.0 +/- 38.0 ng/ml respectively. In the case of urine obtained from rats bearing mammary adenocarcinomas, the mean p65 level was 119.0 +/- 35.9 ng/ml and their serum level was 225.4 +/- 67.5 ng/ml. Sensitivity, specificity and predictive value for serum and urine marker elevation were 78.5, 70.0 and 78.5% respectively. Following in vitro phosphorylation of concentrated urinary proteins, isoelectrofocusing, SDS-PAGE and autoradiography, a phosphorylated form of the 65 kDa protein with a pI of 5.8 was identified in the urine of tumor-bearing rats. This phosphoprotein bound to an antiphosphotyrosine monoclonal antibody and an anti-p65 polyclonal as determined by Western blot analysis. Using the anti-p65 antibodies in an immunoprecipitation procedure, the main radio- and immunoactive band of 65 kDa and two lower mol. wt bands of 50 and 41 kDa, apparently representing degradation products of p65, were identified after in vitro and in vivo phosphorylation of urinary proteins obtained from mammary carcinoma-bearing rats.

Monoclonal antibodies against a 65-kDa tumor-associated phosphoprotein: development and use in cancer detection.

Five hybridoma cell lines secreting monoclonal antibodies (MAbs) to a 65-kDa tumor-associated phosphoprotein (p65) were established. Purified to homogeneity, p65 was used as an immunogen to induce immune response in C57BL/6N mice. Splenocytes were fused with mouse myeloma cells and hybridoma lines were selectively subcloned. A rapid and sensitive sandwich type ELISA, using purified MAbs was established to measure markedly elevated amounts of p65 in sera obtained from both tumor-bearing rats and from cancer patients. The p65 from rat and human sources was added quantitatively to normal sera to construct standard curves. The average level of p65 in normal rat sera was 38 ng/ml +/- 13 ng/ml (mean +/- SD), and in sera from rats bearing mammary adenocarcinomas, the average value was 1005 +/- 140 ng/ml. In normal human sera the mean level of p65 was 34 +/- 35 ng/ml (mean +/- SD) and sera of patients with variety of cancers had an average p65 value of 344 +/- 57 ng/ml. More than 80% of tested sera from adenocarcinoma-bearing rats (20/24) as well as from cancer patients (82/98) had p65 levels elevated two standard deviations above the mean. Overall the assay had a sensitivity of 80.9% and specificity of 85%. The purified IgG1 MAbs, with high titers and strong anti p65 specificities were also used to develop an immunohistochemical method to visualize the expression of p65 in rat tumor tissue sections. The HB2, HF11 and RE6 cell lines have proved to be quite stable in the ability to secrete anti-p65 MAbs.

Potential use of D-glucaric acid derivatives in cancer prevention.

There is now growing evidence from animal models for the possible control of different stages of the carcinogenic process by the beta-glucuronidase inhibitor D-glucaro-1,4-lactone and its precursors such as D-glucaric acid salts, D-glucarates. D-Glucaric acid is a natural, non-toxic compound produced in small amounts by mammals, including humans. It was recently found in some vegetables and fruits. D-Glucaro-1,4-lactone and D-glucarate exhibit potent antiproliferative properties in vivo. Some human subpopulations could have reduced risk of cancer development by ingesting food rich in D-glucaric acid or self-medication with D-glucarates alone or in combination with other chemopreventive agents.

Antiproliferative effect of dietary glucarate on the Sprague-Dawley rat mammary gland.

Dietary glucarate has previously been shown to inhibit chemical carcinogen-induced rat mammary tumorigenesis. It is demonstrated in this paper that in the mammary gland of the female Sprague-Dawley rat, feeding glucarate at a dose of 70 mmol/kg AIN76A diet for 2 weeks beginning at 35 days of age, markedly reduces [3H]thymidine labeling. Specific histochemical staining for beta-glucuronidase is used to show that the glucarate diet fed to rats for 2-4 weeks inhibits beta-glucuronidase activity in the mammary gland and has a marked antiproliferative effect on mammary epithelium. Glucarate may inhibit rat mammary carcinogenesis, in part, by changing the proliferative status of the target organ.

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.