Cycloheximide or puromycin can substitute for PDGF in inducing cellular DNA synthesis in quiescent 3T3 cells.

A brief exposure of quiescent (Go) Swiss 3T3 mouse fibroblasts to inhibitors of protein synthesis can replace platelet-derived growth factor in the stimulation of cellular DNA synthesis. When 3T3 cells, after a 6 hr exposure to either cycloheximide or puromycin, are incubated with platelet-poor plasma, a significant percentage of cells enters DNA synthesis. Either inhibition of protein synthesis, or platelet poor plasma by themselves are totally ineffective.

Regulation of the expression of the SV40 T-antigen coding gene under the control of an rDNA promoter.

We have constructed a hybrid gene in which the SV40 T-antigen coding gene is driven by a mouse rDNA promoter and we have compared its expression to that of an SV40 T-antigen coding gene under the control of its own promoter. The comparison has been carried out in microinjected cells, in transfected cells, and in stable cell lines carrying the respective T-antigen coding genes in an integrated form. These cell lines were derived from ts AF8 cells, a mutant which is temperature sensitive for RNA polymerase II activity.

Expression of cell cycle-dependent genes in young and senescent WI-38 fibroblasts.

We studied the expression of 11 cell cycle-dependent genes in senescent WI-38 fibroblasts and compared the results to those obtained in WI-38 cells from early passages (young cells). Every gene we examined is expressed in the senescent cells at levels similar to those in the young cells, including two genes maximally expressed at the G1/S phase boundary--genes for thymidine kinase and histone H3. The results clearly show that senescent, noncycling WI-38 cells are not similar to quiescent cells.

Altered expression of G1-specific genes in human malignant myeloid cells.

We have studied the expression of cell-cycle genes specific to the G1 (2A9, 2F1, 4F1, c-myc) and S (histone H3) phases of the cell cycle in normal and malignant human myeloid cycling cells. The levels of expression were determined by measuring the amounts of specific RNA in blot hybridization assays. Levels of expression of the G1 genes were compared to the level of expression of the S-phase-specific H3 gene.

Molecular biology of the cell cycle.

Genes and cDNA clones have been identified in animal cells that are cell cycle-regulated, i.e. they are preferentially expressed in a phase of the cell cycle.

Co-operation between the p53 protein tumor antigen and platelet-poor plasma in the induction of cellular DNA synthesis.

Plasmids containing DNA sequences coding for p53 were microinjected into quiescent Swiss 3T3 cells. Three constructs were used, carrying either the whole gene sequence, a full-length cDNA, or a hybrid between the gene and the cDNA. All of them stimulated DNA synthesis when cells were incubated with platelet-poor plasma (PPP) following injection.

Effect of interleukin-2 on the expression of cell cycle genes in human T lymphocytes.

We have studied the expression of seven cell cycle-dependent genes in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells, in macrophage-depleted cultures and in macrophage-depleted cultures plus Interleukin-2 (IL-2). The expression of all seven genes is increased in PHA stimulated peripheral cells. Only two (2F1 and the IL-2 receptor) are increased in PHA-stimulated macrophage depleted cultures.

Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2.

The effect of cycloheximide on the expression of cell cycle dependent genes.

We have investigated the inducibility of several cell cycle-dependent genes (plus control sequences, not expressed in a cell cycle-dependent manner) in the presence of cycloheximide, an inhibitor of protein synthesis. The genes studied include: 1) five cDNA clones that are preferentially expressed in the G1 phase of the cell cycle: KC-1, JE-3, 2F1, 4F1 and 2A9; 2) one gene preferentially expressed in late G1/S phase: histone H3; and 3) the cell cycle-dependent oncogene p53. All the genes studied are induced by serum even in the presence of cycloheximide.

Expression of the p53 protein during the cell cycle of human peripheral blood lymphocytes.

We have investigated the role of the cellular p53 protein in the induction of growth in size and cell DNA replication in human peripheral blood lymphocytes (PBL) and in monocyte/macrophage-depleted lymphocyte (MDL) cultures stimulated with phytohemagglutinin (PHA). Our results show that in human lymphocytes exposed to PHA, the induction of p53 protein synthesis and accumulation correlates with the extent of cellular DNA replication, rather than with growth in size. Moreover, the induction of p53 is dependent on the presence of the T-cell mitogen, Interleukin-2.

Expression of cell-cycle-dependent genes in phytohemagglutinin-stimulated human lymphocytes.

We have investigated the expression of certain cell-cycle-dependent genes in human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA). The genes studied had been previously identified as cell-cycle dependent in other cell types from different species and were induced by different mitogens. One of these genes (2F1) and the gene for the interleukin 2 receptor were induced by PHA even in cultures partially depleted of accessory cells where the lymphocytes grew in size but failed to enter S phase.

Cell-cycle-specific genes differentially expressed in human leukemias.

Three cDNA clones isolated from Syrian hamster cells (p4F1, p2F1, and p2A9) contain sequences that are preferentially expressed in the G1 phase of the cell cycle. The expression of these sequences was investigated in human peripheral blood cells from normal individuals and from patients with leukemia. The expression of p4F1 and p2F1 is clearly dependent on the cell cycle in peripheral blood mononuclear cells stimulated to proliferate with phytohemagglutinin; the p2A9 sequences cannot be clearly detected in human lymphocytes but are expressed in a cell-cycle-dependent manner in human diploid fibroblasts (WI-38).

Microinjected c-myc as a competence factor.

While a number of oncogenes are expressed in a cell cycle-dependent manner, their role in the control of cell proliferation can only be established by a direct functional assay. The c-myc protein, upon microinjection into nuclei of quiescent Swiss 3T3 cells, cooperated with platelet-poor plasma in the stimulation of cellular DNA synthesis. This suggests that c-myc protein, like platelet-derived growth factor (PDGF), may act as a competence factor in the cell cycle to promote the progression of cells to S phase.

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.

Expression of thymidine kinase and dihydrofolate reductase genes in mammalian ts mutants of the cell cycle.

Thymidine kinase and dihydrofolate reductase mRNA levels and enzyme activities were determined in two temperature-sensitive cell lines, tsAF8 and ts13, that growth arrest in the G1 phase of the cell cycle at the restrictive temperature. The levels of thymidine kinase mRNA and enzyme activity increased markedly in both cell lines serum stimulated from quiescence at the permissive temperature. At the nonpermissive temperature, the levels of thymidine kinase mRNA and enzyme activity remain at the low levels of quiescent G0 cells.

Effect of butyrate on the expression of microinjected or transfected genes.

We have studied the effect of sodium n-butyrate on the expression of specific genes. For this purpose, tk-ts13 cells (a thymidine kinase-deficient mutant originating from Syrian hamster cells) were microinjected or transfected with pC2, a plasmid containing the entire SV40 genome and the herpes simplex virus thymidine kinase gene (HSV-TK), cloned in pBR322. As a measure of the expression of these two genes, one of which is spliced (SV40) and the other one (HSV-TK) which is not, we have taken the protein levels (amount of T antigen for SV40 and incorporation of [3H]thymidine for HSV-TK) and the levels of RNA (by dot blot hybridization).

Microinjected ras family oncogenes stimulate DNA synthesis in quiescent mammalian cells.

Oncogenes of the ras family stimulate DNA synthesis when microinjected into quiescent mouse and hamster fibroblasts, as detected by in situ autoradiography. The molecularly cloned genomes of Harvey and Kirsten sarcoma viruses, the cloned Harvey ras gene, and the product of the v-ras gene, the p21v-rasH protein, stimulate DNA synthesis in quiescent cells. This stimulation is comparable to the stimulatory activity of the microinjected SV40 T-antigen-coding gene.

Cell-cycle-specific cDNAs from mammalian cells temperature sensitive for growth.

A library of double-stranded cDNA was constructed from ts13 cells, a G1-specific temperature-sensitive hamster cell line. The cDNAs, cloned into pBR322, were prepared from poly(A)+ mRNA isolated from ts13 cells 6 hr after serum stimulation at the permissive temperature of 34 degrees C. Differential screening of the library with G1-specific and G0-specific single-stranded cDNA probes prepared from the same cells identified five cDNA clones whose sequences were preferentially expressed in G1.

Expression of histone genes in a G1-specific temperature-sensitive mutant of the cell cycle.

The expression of genes coding for the four core histones (H2A, H2B, H3, and H4) was studied in tsAF8 cells. These baby hamster kidney-derived cells are a temperature-sensitive (ts) mutant of the cell cycle that arrest in G1 at the restrictive temperature. When serum-deprived tsAF8 cells are stimulated with serum, they enter the S phase at the permissive temperature of 34 degrees C, but are blocked in G1 at the nonpermissive temperature of 39.

Transforming potential of deletion mutants of the SV40 T antigen coding gene in Syrian hamster cells.

Thymidine kinase-deficient syrian hamster cells were cotransfected with recombinant plasmids containing the thymidine kinase (TK) gene of Herpes Simplex Virus Type 1, and either intact or partially deleted SV40 T antigen-coding genes. The transformants were selected by their ability to grow in gHAT medium. After selection and cloning, the TK-positive transformants that also expressed T antigen were tested for the extent of their transformation with respect to a number of characteristics, which included saturation density, ability to grow in soft agar, resistance to butyrate and to dibutyryl-cAMP, and plating efficiency.

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.

Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants.

Microinjection of RNA polymerase II corrects the temperature-sensitive defect of tsAF8 cells.

tsAF8 cells are a temperature-sensitive (ts) mutant of BHK cells that arrest in the G1 phase of the cell cycle at the non-permissive temperature of 40.6 degrees C. Previous reports had suggested that the temperature-sensitivity of these cells was based on a defect in either the synthesis, assembly or turnover of RNA polymerase II.

Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA.

We have used an antibody against RNA polymerase I to investigate the role of rRNA synthesis and/or accumulation in the control of cell proliferation. The antibody was microinjected directly into the nuclei of quiescent Swiss 3T3 cells that were subsequently stimulated with serum. Under the experimental conditions used, the microinjection of the antibody against RNA polymerase I (RNA pol I) caused a 50-70% decrease in nucleolar RNA synthesis that lasted for at least 17 h, a greater than 90% inhibition in the accumulation of nucleolar RNA, and a 70% inhibition in the accumulation of total cellular RNA.