Post-translational regulation of the 54K cellular tumor antigen in normal and transformed cells.

The 54K cellular tumor antigen has been translated in vitro, using messenger ribonucleic acids from simian virus 40 (SV40)-transformed cells or 3T3 cells. The in vitro 54K product could be immunoprecipitated with SV40 tumor serum and had a peptide map that was similar, but not identical, to the in vivo product. The levels of this 54K protein in SV3T3 cells were significantly higher than those detected in 3T3 cells (D. I. H. Linzer, W. Maltzman, and A. J. Levine, Virology 98:308-318, 1979). In spite of this, the levels of translatable 54K messenger ribonucleic acid from 3T3 and SV3T3 cells were roughly equivalent or often greater in 3T3 cells. Pulse-chase experiments with the 54K protein from 3T3 or SV3T3 cells demonstrated that this protein, once synthesized, was rapidly degraded in 3T3 cells but was extremely stable in SV3T3 cells. Similarly, in an SV40 tsA-transformed cell line, temperature sensitive for the SV40 T-antigen, the 54K protein was rapidly turned over at the nonpermissive temperature and stable at the permissive temperature, whereas the levels of translatable 54K messenger ribonucleic acid at each temperature were roughly equal. These results demonstrate a post-translational regulation of the 54K cellular tumor antigen and suggest that this control is mediated by the SV40 large T-antigen.     

Overlapping of normal and malignant mouse cells in pure and mixed cultures

Cultured malignant mouse melanoma cells and mouse ascites carcinoma cells, after 24 h incubation with normal mouse cells or with each other, had much higher homologous overlap indices than those recorded from pure cultures of each cell type. The effect was particularly evident in ascites cells. Normal cells of mice of different ages showed varying responses in overlap frequency when incubated with malignant cells. Conditioned medium from each malignant cell type invoked increased overlapping of cells of the heterologous malignant type but did not affect homologous cells. The effects of whole conditioned medium on heterologous malignant cells showed a graded reduction after dilution of the medium and also occurred after dialysis. A dialysable chemical factor or factors, produced by the cells, is postulated.     

The effect of procarbazine on the chromosomes of normal and malignant mouse cells.

The effects of the cancer drug methylhydrazine derivative procarbazine (PC) on the chromosomes of normal and malignant cells in the mouse strains Swiss Albino, CF1, and BDF1 have been analyzed. PC broke chromosome in the hypotetraploid Ehrlich and the diploid P388 ascites tumors. Practically all breaks seemed to be of the G2 type and, as far as could be determined, took place in the quinacrine-dark or the corresponding Giemsa-light regions. The vast majority of the broken ends had rejoined to form chromatid translocations. No increase in chromosome breaks were seen in any of the normal tissues treated in vivo: spleen, bone marrow, spermatocytes, or spermatogonia (in this tissue the cells were not counted). The chromosomes of the Ehrlich ascites tumor have been studied by means of both Q-banding and G-banding. Very few, if any, chromosomes seem to have an exact homologue or to correspond to any of the normal mouse chromosomes.     

Quantitation and potential function of nucleolar phosphoprotein pp 105 in mouse tumor cells, embryonic cells and normal tissues

1. Nucleolar phosphoprotein pp 105 was determined in various mouse cell and tissue extracts using a highly sensitive ELISA. The results indicate that the highest relative amounts of pp 105 correlate with cells and tissues of high growth rate such as tumor cell lines, solid tumors and embryonic tissues. 2. The specific phosphorylation of pp 105 was compared in a 1 min endogenous phosphorylation assay with native cell and tissue extracts. 3. The mitogenic activity of highly purified pp 105 was demonstrated in cultures of resting mouse embryonic cells and mouse thymocytes.     

Distinct requirements of adenovirus E1b55K protein for degradation of cellular substrates

The E1b55K and E4orf6 proteins of adenovirus type 5 (Ad5) assemble into a complex together with cellular proteins including cullin 5, elongins B and C, and Rbx1. This complex possesses E3 ubiquitin ligase activity and targets cellular proteins for proteasome-mediated degradation. The ligase activity has been suggested to be responsible for all functions of E1b55K/E4orf6, including promoting efficient viral DNA replication, preventing a cellular DNA damage response, and stimulating late viral mRNA nuclear export and late protein synthesis. The known cellular substrates for degradation by E1b55K/E4orf6 are the Mre11/Rad50/Nbs1 DNA repair complex, the tumor suppressor p53, and DNA ligase IV. Here we show that the degradation of individual targets can occur independently of other substrates. Furthermore, we identify separation-of-function mutant forms of E1b55K that can distinguish substrates for binding and degradation. Our results identify distinct regions of E1b55K that are involved in substrate recognition but also imply that there are additional requirements beyond protein association. These mutant proteins will facilitate the determination of the relevance of specific substrates to the functions of E1b55K in promoting infection and inactivating host defenses.     

Quantitation and tissue localization of the cellular retinoic acid-binding protein

The distribution of the cellular retinoic acid-binding protein (CRABP) in some rat tissues has been determined, and the protein has been localized by immunocytochemical techniques in sections from rat testis. In the testis CRABP was found in the seminiferous tubuli with Sertoli cells and the spermatogonia most intensely stained. All other cells of the germinal epithelium appeared largely devoid of CRABP. By use of an enzyme-linked immunosorbent assay CRABP was quantitatively estimated in several tissues and the highest levels were found in testis and eye. Comparisons of the tissue levels of CRABP and of the cellular retinol-binding protein (CRBP) did not reveal any apparent correlation.     

Distinction between malignant L cells and normal mouse fibroblasts by rosette formation with sheep red blood cells.

Murine L cell fibroblasts, and derivatives were found to rosette with sheep red blood cells (SRBC). Primary fibroblast explants from the parent murine strain, C3H, did not possess this potential. No rosettes were observed with primary fibroblast explants from C57BL and B10Br mice, with a human fetal lung fibroblast, with baby hamster fibroblasts or their polyoma transormed derivative, or with a cell line, 1T-22, derived from BALB/c mice. Hybridization of 1T-22 and L cells, by Sendai virus-mediated cell fusion, suppressed the rosette potential of the L cell parent. The receptor for SRBC on L cells appears to result from the expression of a recessive characteristic.     

The amount of a specific cellular protein (p53) is a correlate of differentiation in embryonal carcinoma cells

A specific cellular protein of molecular weight of 53–55,000 (p53) has been shown to be induced in all SV40 transformed cells. A similar protein has also been shown to be present in embryonal carcinoma cells and in midgestation murine embryo primary cells, which are not infected by SV40. In embryo cell primaries the amount of the protein was shown to decrease with the increase in the stage of embryo development. As differentiation or decrease in cell growth rate can account for this, and since the growth rate of embryo primary cells cannot be measured, we chose to investigate various embryonal carcinoma cells. We report that the p53 is present in a pluripotent embryonal carcinoma cell OTT6050, and in its differentiated parietal endoderm derivative, PYS-2 cells. The amount of p53 is higher in the undifferentiated EC stem cells than in the differentiated PYS-2 (parietal endoderm) cells. The amount of the protein decreases in F9 embryonal carcinoma cells induced to differentiate to a parietal endoderm cell type by treatment with retinoic acid, as it does following spontaneous differentiation of OTT6050 EC cells. To determine if a change in growth rate, rather than differentiation, might acount for the diminished levels of this protein, the amount ofp53 was measured in growing and in growth arrested cell populations. When the growth rate of F9 cells was reduced by treatment with 8-bromocyclic AMP there was no change in the amount of p53. The half life of the p53 was compared in the undifferentiated and the differentiated cell types to determine if a change in stability might account, in part, for the altered levels of this protein. The p53 is found to be most stable in the SV40 transformed established clonal cells. It is less stable in the fibroblast clonal cells which were not transformed by SV40. The results of these experiments indicate that a decrease in the amount of p53 primarily correlates with differentiation in the embryonal carcinoma cell lines studied and not with cell growth rate. Furthermore, the decrease appears to be related (in part) to the decreased stability of the p53.     

Identification of a prominent nuclear protein associated with proliferation of normal and malignant B cells

Experiments were undertaken to identify nuclear proteins that might be involved in regulation of the mitogenic process in B lymphocytes. Murine splenic B lymphocytes were purified and cultured with anti-Ig insolubilized onto Sepharose (anti-Ig/Sepharose) for 16 hr and labeled with [35S]methionine. Nuclei were isolated and the nuclear proteins were analyzed by two-dimensional gel electrophoresis. Anti-Ig/Sepharose induced a prominent increase in the synthesis and abundance of a 40 kDa/pI 5 nuclear protein (p40/pI-5). Inhibition of anti-Ig/Sepharose-induced mitogenesis by pretreatment of the cells with phorbol-12-myristate-13-acetate was associated with a specific inhibition (63%) of p40/pI-5. Subcellular fractionation experiments showed that p40/pI-5 is not detected in the soluble fraction of resting or activated B cells, indicating that this protein is located exclusively in the nucleus. Analysis of the expression of p40/pI-5 relative the cell cycle showed that the synthesis of this protein was increased during G1 phase and gradually reduced during S phase of the cell cycle. Abundant amounts of p40/pI-5 were also found in the rapidly proliferating B lymphoma cells, WEHI-231, and growth arrest of these cells by anti-mu was found to be associated with a marked inhibition (68%) of this protein. Taken collectively these results suggest that the nuclear protein p40/pI-5 may have an important role in regulation of the proliferation of normal and malignant B lymphocytes.     

A distinctive phospholipid-stimulated protein kinase of normal and malignant murine hemopoietic cells

This report describes the activity of a novel phospholipid-stimulated protein kinase from mouse DA-1 leukemic cells. The kinase was activated by phosphatidylglycerol or phosphatidylinositol. Phospholipid-stimulated protein phosphorylation occurred in the presence of Mn2+ or Mg2+; kinase activity was greater with Mg2+ than with Mn2+ from 4 to 10 mM, although at lower divalent cation concentrations Mn2+ was preferred. A Mr 75,500-77,000 endogenous protein doublet and a Mr 42,000 endogenous protein were phosphorylated in whole cell extracts under these conditions. These substrates contrasted with those identified under protein kinase C conditions. Of the exogenous proteins tested, phospholipid-stimulated phosphorylation was highest with histone H2B followed by other histones. In addition to DA-1 cells, phospholipid-stimulated protein kinase also was detected in high levels in normal mouse spleen, marrow, and kidney but not detectable in brain extracts. The phosphatidylglycerol-stimulated kinase was separated from protein kinase C by anion-exchange chromatography on DEAE-Sephacel, from which it eluted at 0.2 to 0.3 M NaCl. Physiological dissociation of the two types of kinase activity was demonstrated by down regulation of protein kinase C over 24 h by phorbol 12-myristate 13-acetic acid. Under these conditions phosphatidylglycerol kinase activity and subcellular distribution were unaffected. Thus, phosphatidylglycerol-stimulated kinase was detectable in both normal and malignant cells and contrasted with, and was separable from, protein kinase C in numerous respects. Phosphatidylglycerol-stimulated protein kinase basic biochemistry and physiological roles are topics worthy of further investigation.     

An abortus with a normal/trisomy 16 mosaicism: instability of trisomic cells in vitro.

A 46,XX/47,XX,+16 mosaicism was demonstrated in cultured chorionic cells obtained from an abortion material. By tracing polymorphic fluorescent chromosome markers in the abortus and the parents, the karyotypically normal component was confirmed to be non-maternal in origin. The trisomic cells showed a growth disadvantage, and were overtaken by the normal cells in prolonged cultivation.     

Expression of malignancy in hybrids between normal and malignant cells.

Somatic cell hybrids between either normal human fibroblasts, phenotypically normal mouse fibroblasts or mouse peritoneal macrophages and HT1080 human diploid fibrosarcoma cells were studied for their ability to form tumors in nude mice. The results of this study indicate that tumorigenic behavior is expressed as a dominant trait in both human-human and mouse-human hybrid cells.     

Acetylcholinesterase in normal and malignant human cells

Acetylcholinesterase (AChE) activity was determined in normal and malignant human cell lines by histochemical methods. In normal human fibroblasts, no AChE activity could be demonstrated by any histochemical technique or substrate. Enzymic activity was observed in HT-1080 human fibrosarcoma cells, RD 2 human rhabdomyosarcoma cells, and SW 311 human colon carcinoma cells. Activity was localized around the nuclear envelope, in the cytoplasm and associated with the cortical region of most cells. The specificity of the reaction was shown through the use of specific cholinesterase inhibitors.     

Effect of benz(a)pyrene on a monolayer culture of normal and malignant mouse liver cells]

Both the cells of monolayer culture of mouse embryonic liver and that of highly malignant hepatoma 22A transplanted for 20 years actively metabolized the carcinogenic hydrocarbon benz(a)pyrene and were highly sensitive to iits toxic action. Since hepatic tissue was resistant in vivo to carcinogenic carbohydrates it is suggested that the resistance depended on factors acting at the organ or the organism but not as the cellular level. The mechanism of retention of hepatoma 22A sensitivity to the toxic action of benz(a)pyrene is also discussed.