Levy Kopelovich

Human skin fibroblasts from individuals genetically predisposed to cancer are sensitive to an SV40-induced T antigen display and transformation

In the present study we have used a viral probe to determine the genetic susceptibility of fibroblastic cell strains derived from individuals with hereditary adenomatosis of the colon and rectum (ACR), an autosomal dominant trait. This report shows an increased sensitivity of apparently karyologically‐normal diploid skin fibroblasts from ACR individuals to an SV40‐induced T antigen display and transformation. None of the SV40‐transformed cells grew as tumors in athymic mice and they all appeared to have a finite life span. The results suggest that the induction of T antigen positive cells by SV40 may be used as a marker of cancer risk in this cell system.

Tumor Promoter Induces Loss of Anchorage Dependence in Human Skin Fibroblasts from Individuals Genetically Predisposed to Cancer

To examine the role of germinal mutation in transformation by phorbol esters, we studied the induction of anchorage-independent variants of mutant human diploid fibroblasts derived from normal-appearing skin of individuals with hereditary adenomatosis of the colon and rectum (ACR). Liquid cultures were chronically exposed to 12-0-tetradecanoyl phorbol-13-acetate (TPA), then plated in agar and injected subcutaneously into athymic mice. Cultured ACR cells showed an unusual biphasic dose response to TPA. Colony-forming cells in agar were obtained at a frequency of about 5 x 10(-5). They did not, however, seem to increase in frequency during subsequent passages in liquid cultures continuously exposed to TPA. The isolated anchorage-transformed clones showed an altered clonal morphology and a considerable increase in cloning efficiency in liquid cultures and agar. The results suggest that ACR cells may be used to screen for potential tumor promoters in our environment.

Regulation of aspartate amino-transferase isozymes by glyceraldehyde-3-phosphate

Abstract This review concerns our work on glyceraldehyde-3-P as a time-dependent inhibitor of rat liver aspartate aminotransferase isozymes, and the possibility that glyceraldehyde-3-P may function as a rapidly acting regulator of aspartate aminotransferase. The d -isomer and the dl -racemate of glyceraldehyde-3-P were equally effective for each isozyme. Study of several glycolytic intermediates indicated that the conjoint presence of the free aldehyde and the phosphoryl residue was necessary for inhibition. Maximum inhibition of the anionic isozyme occurred at pH values from 8.4 to 10.3; the cationic isozyme was optimally inhibited at pH 7.4. Keto acid substrates decreased the inhibition, whereas amino acid substrates accentuated it. Inhibition of the cationic isozyme was completely competitive with respect to α-ketoglutarate ( K i = 0.98 m m ) and oxaloacetate (Ki not measurable), and completely noncompetitive with respect to l -aspartate ( K i = 0.084 m m ) and l -glutamate ( K i = 0.11 m m ). Inhibition of the anionic isozyme was mixed partially competitive-partially noncompetitive with respect to α-ketoglutarate ( K i = 1.9 m m ) and oxaloacetate ( K i = 1.5 m m ) and partially noncompetitive with respect to l -aspartate ( K i = 0.39 m m ) and l -glutamate ( K i = 0.57 m m ). These data suggest that both keto acids bind to the isozymes at a single site and compete with glyceraldehyde-3-P for that site, whereas the amino acids bind to a site other than the one for which keto acids and glyceraldehyde-3-P compete. Homologues of glyceraldehyde-3-P were also investigated. Ribose-5-P, fructose-6-P and glucose-6-P did not inhibit either isozyme since they exist as the internal hemiacetals. d -erythrose-4-P was a time-dependent inhibitor of both isozymes. Inhibition was completely competitive with respect to α-ketoglutarate, K i = 3.08 m m and 1.4 m m for the anionic and cationic isozymes, respectively, and was completely noncompetitive with respect to l -aspartate, K i = 0.334 m m and 0.135 m m for the anionic and cationic isozymes, respectively. Inhibition by glycolaldehyde-P was not time-dependent and was completely competitive with respect to α-ketoglutarate and uncompetitive with respect to l -aspartate for both isozymes. The Ki values were 0.77 m m and 1.01 m m for the anionic and cationic isozymes, respectively. We propose that glyceraldehyde-3-P and its homologues inhibit aspartate aminotransferase isozymes by forming a Schiff base with one of the ϵ-amino groups of lysine at the enzymically active site. Aspartate would potentiate inhibition by converting the enzyme to the pyridoxamine form, thereby exposing a second ϵ-amino lysyl group which would react with the inhibitor. Evidence for a Schiff base was obtained by NaBH4 reduction of apoisozymes in the presence of dl -glyceraldehyde-3-P. This prevented restoration of activity upon addition of pyridoxal-5′-P. It seems likely that the divalent phosphoryl group on the inhibitor molecule is involved in the competition at the keto acid binding site. Our results suggest the possibility that glyceraldehyde-3-P may be implicated in the regulation, in vivo, of gluconeogenesis as well as other metabolic pathways by affecting the activity of the isozymes of aspartate aminotransferase.

Neoplastic Transformation of Human Mutant Cells by a Tumor Promoter

Neoplastic transformation is a multi-phase process apparently caused by carcinogens and subject to the influence of promoters. The naturally occurring phorbol esters such as 12-0-tetradecanoyl phorbol-13-acetate (TPA) are potent tumor promoting agents. We have studied the effects of TPA on human mutant fibroblast cell.strains derived from Individuals with hereditary adenomatosis of the colon and rectum (ACR), an autosomal dominant trait. We have previously demonstrated in these fibroblasts abnormal phenotypic expressions which often appear in transformed cells. In these studies, we have assumed that the ACR cell exists in an ‘initiated state’ due to a dominant mutation and that expression of the malignant state might only require treatment with a promoting agent. This simple experimental protocol provided a novel system for the study of cancer promotion in vitro. We have now demonstrated the growth in vivo and growth properties in vitro of human mutant cells exposed to TPA alone.

A Nontumorigenic (Mouse × Human) Cell Hybrid Inhibits Tumorigenicity of Its Malignant Mouse Parent Cell and Unrelated Human Tumor Cells in vivo

This report describes the modulation of malignant through coinoculation in vivo of a malignant mouse (A9) cell line, and a phenotypically stable, nontumorigenic hybrid clone derived from it by fusion with a transformed mutant human cell. Thus, the tumorigenicity of the A9 cells was suppressed by the hybrid cells, but not by the unrelated normal cells. The hybrid cells also inhibited tumor development of unrelated human cancer cells.

The Suppression of Tumorigenicity in Human X Mouse Cell Hybrids

We have studied the expression of transformation-related parameters in tertiary hybrid clones from an apparently transformed human cell strain (ME) and a malignant mouse cell line (A9). The derivation of these hybrid clones, chromosome analysis, and tumorigenic potential have been reported elsewhere (1). In the present work, the serum sensitivity, saturation density, cloning efficiency on plastic surfaces and in semisolid medium, survival in aggregate form, concanavalin A agglutinability, sugar uptake, and secretion of plasminogen activator in 4 tertiary hybrid clones and in both parent cells have been determined. While, in general, the extent to which these transformation-related traits were expressed in the hybrid clones closely resembled the malignant A9 parent cell, all hybrids displayed partial to complete suppression of tumorigenicity. These results suggest no apparent relationship between the extent of quality of transformation and tumorigenicity in these cell hybrids.

Pyruvate dehydrogenase, the citrate condensing enzyme and the utilization of 14 C-labeled lactate, pyruvate and alanine by slices of lactating mammary gland and adenocarcinoma of mouse mammary gland.

Summary The utilization of 14C-labeled lactate, pyruvate and alanine by tissue slices of lactating mouse mammary gland and adenocarcinoma of the mouse mammary gland was investigated. In the absence of glucose, rates of 14CO2 formation and fatty acid synthesis from these substrates in the mammary adenocarcinoma were considerably lower than those in the lactating mammary gland, regardless of the position of the labeled carbon. In the lactating mammary gland, addition of glucose caused a considerable decline in 14CO2 formation from the second and third carbons of lactate, pyruvate and alanine, while it significantly stimulated their incorporation into fatty acids. Glucose also stimulated 14CO2 formation from the first carbon of these substrates. In the mammary adenocarcinoma, however, 14CO2 formation from the first carbon of lactate and pyruvate decreased upon glucose addition, while that from the second and third carbons was almost unaffected. The incorporation into fatty acids of the second and third carbons from these substrates in the neoplasm was only slightly stimulated by glucose. Lactate uptake by lactating mammary gland was twice that observed in the mammary adenocarcinoma, but amino acid formation from this substrate was significantly higher in the neoplasm. The synthesis of glutamine, however appears to be defective in the mammary adenocarcinoma. The data indicated considerably lower levels of pyruvate dehydrogenase (EC 1.2.4.1) and citrate condensing enzyme (EC 4.1.3.7), and possibly higher acetyl-CoA deacylase (EC 3.1.2.1) activities in the tumor than in the lactating mammary gland. The significance of this finding in relation to the entry of 3-carbon glycolytic intermediates into the Krebs cycle in both the lactating mammary gland and the mammary adenocarcinoma is discussed.

The Utilization of 14C-Labeled Lactate, Pyruvate and Alanine by Prelactating Tissues and Hyperplastic Alveolar Nodule Outgrowths

Summary The utilization of 14C-labeled lactate, pyruvate and alanine by specimens of prelactating tissue taken from pregnant mice and hyperplastic alveolar nodule outgrowths from C3H mice was investigated. In the absence of glucose, CO2 production and fatty acid synthesis from these substrates was higher in the prelactating mammary gland than in the hyperplastic alveolar nodule outgrowths. Addition of glucose effected a decrease in CO2 production from all 3 carbons, but caused an increase in fatty acid formation from the second and third carbons of these substrates. This effect by glucose was proportionately similar in both tissues. Lactate uptake by the prelactating mammary gland was about 60% higher than that by the hyperplastic alveolar nodule outgrowths, but amino acid formation from this substrate was markedly higher in the latter tissue. The activity of the citrate condensing enzyme (EC 4.1.3.7) was considerably lower in the hyperplastic alveolar nodule outgrowths than in the prelactating mammary tissue. An enzymatic alteration at this site would slow entry of 3-carbon glycolytic intermediates into the Krebs cycle and may represent a prime metabolic lesion in the development of the hyperplastic alveolar nodule outgrowths.