Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein

We have identified a cDNA whose sequence is preferentially expressed when quiescent fibroblasts are stimulated to proliferate. The steady-state levels of the mRNA corresponding to this clone, called 2A9, are increased by serum, platelet-derived growth factor, and epidermal growth factor, but not by insulin or platelet-poor plasma. mRNA levels of 2A9 are also increased in human acute myeloid leukemia. The 2A9 cDNA has been molecularly cloned from an Okayama-Berg library, and its complete nucleotide sequence has been determined. It has an open reading frame of 270 nucleotides, which has a 55% homology with the coding sequence of the beta-subunit of the S-100 protein, a calcium-binding protein that belongs (like calmodulin and the vitamin D-dependent intestinal calcium-binding protein) to the family of calcium-modulated proteins and is found in abundance in several human tumors, including melanoma. The S-100 protein and the deduced aminoacid sequence of 2A9 are also partially homologous to the small subunit of a protein complex that serves as a cellular substrate to tyrosine kinase. The partial homology of 2A9 (whose RNA is inducible by growth factors and is overexpressed in human acute myeloid leukemias) to the S-100 protein, other calcium-modulated proteins, and the subunit of a substrate for tyrosine kinase, is particularly interesting in view of the role attributed to calcium and tyrosine kinases in the regulation of cell proliferation.     

Induction of cellular DNA synthesis by a simian virus 40 mutant defective in nuclear transport of T antigen.

The simian virus 40 (SV40) (cT)-3 mutant [SV40(cT)-3], which is defective in nuclear transport of T antigen, was utilized to determine whether cellular DNA synthesis can be stimulated by SV40 in the absence of detectable nuclear T antigen. Cellular DNA synthesis was examined in the temperature-sensitive cell cycle mutants, BHK ts13 and BHK tsAF8, after microinjection of quiescent cells with plasmid DNA containing cloned copies of wild-type SV40 or SV40(cT)-3. The efficiency of induction of cellular DNA synthesis was identical for both wild-type SV40 and SV40(cT)-3 in both cell lines. The results suggest that cell surface-associated T antigen, either alone or possibly in combination with minimal amounts of nuclear T antigen below our limit of detection, is able to stimulate cellular DNA synthesis.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

Changes in the G0 state of WI-38 fibroblasts at different times after confluence

Some events in the prereplicative phase of WI-38 human diploid fibroblasts stimulated to proliferate are found to be a function of the length of time the cells have been quiescent. At 5 days after plating, when the cells first become confluent, the prereplicative phase upon stimulation by a nutritional change is relatively short, DNA synthesis begins at 8 h after stimulation, and there is no increase in chromatin template activity. At 9 days after plating the prereplicative phase of stimulated cells is lengthened to 14 h and there is an increase in chromatin template activity within 1 h of stimulation. Finally, in 18-day cells, the prereplicative phase is lengthened even further to 20 h, and there is a lag after stimulation before the increase in chromatin template activity. It is proposed that confluent WI-38 cells initially arrest in G 1, subsequently pass into G 0, and continue to go deeper into G 0 as they remain quiescent.     

Uptake of radioactive thymidine and cytidine by Ehrlich ascites tumor cells in different stages of growth

Summary In Strong A female mice, the Ehrlich ascites tumor inoculated into the peritoneal cavity grows exponentially for the first 7 days with a doubling time of about 36 hours. The tumor enters then into a late stage during which the number of tumor cells in the peritoneal cavity does not increase. The uptake of intraperitoneally injected thymidine decreases from the exponential to the late stage, mostly because of a decrease in the fraction of cells in DNA synthesis. During the exponential phase, the uptake of thymidine is a function of the amount of radioactive thymidine injected per tumor cell, the utilization decreasing with increasing cell dose. The uptake of intraperitoneally injected cytidine decreases slightly with time after inoculation although the fraction of tumor cells in RNA synthesis remains constant.Dedicated to Professor Friedrich Wassermann with admiration and affection on the occasion of his 80th birthday.This investigation was supported by U.S.P.H.S. Grant CA-05667. The author is a U.S.P.H.S. Research Career Development Awardee.     

The role of the IGF-I receptor in the growth and transformation of mammalian cells

Abstract. Recent developments in the molecular biology of the insulin-like growth factor I (IGF-I) receptor have clarified its role in cellular growth and transformation. Although cells homozygous for a targeted disruption of the IGF-I receptor genes can grow in serum-supplemented medium, the IGF-I receptor is required for optimal growth, and is required equally in all phases of the cell cycle. The receptor plays an even more stringent role in cellular transformation and tumorigenicity, which seem to be dependent on its normal expression in several cell types. The expression of both the IGF-I receptor and its ligands is regulated by other growth factors (especially PDGF and EGF), by oncogenes (like SV40 T antigen and c-myb) and by tumour suppressor genes (like WT1 and RB). The picture emerging from these studies is that several transforming agents may exert their growth promoting effects through the direct or indirect activation of the IGF autocrine loop.