Effects of the tumor promoter TPA on the induction of DNA synthesis in normal and RSV-transformed rat fibroblasts

Induction of DNA synthesis by the tumor promoter tetradecanoyl phorbol acetate (TPA) was studied in a line of cultured rat fibroblasts (Rat-1) and their ffRous sarcoma virus-transformed derivative (Rat-1(RSV)). Following serum deprivation for 54 h to achieve quiescene, semiconservative DNA replication was measured by incubation of cells in BrdUrd and FdUrd after serum stimulation in the presence or absence of TPA. Optimal concentrations of TPA (0.1–0.5 μg/ ml) in serum-free medium induced a small increase (10–15%) in the amount of DNA made over a 30-h period in both Rat-1 and Rat-1 (RSV) cells. When Rat-1 cells were stimulated by a 4-h serum pulse, 30% of the DNA was replicated by 30 h. If the serum pulse was follwed by TPA addition, 702% DNA replication wass observed. If the serum pulse was preceded by TPA addition, the onset of DNA synthesis waas delayed by several hous, but stimulation of DNA synthesis occurred. In contrast, the Rat-1 (RSV) cells did not show an increase in DNA synthesis induced by TPA in similar protocols, but the serum-induced onset on DNA synthesis was delayed by several hours in the presence of TPA. Therefore, TPA acts as a co-inducer of DNA synthesis in the Rat-1 but not in the Rat-(RSV) cells. The parent alcohol, phorbol, was inactive in Rat-1 cells, but delayed the onset of DNA synthesis in the Rat(RSV) cells. We conclude that the co-inducing and delaying activities of TPA on DNA synthesis appear to be distinct and to act at different points in the G₁ phase of the cell cycle.     

Calcium compartments and fluxes are affected by the src gene product of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus

Total cellular calcium content (determined by atomic absorption spectrometry) of Rat-1 cells transformed by temperature-sensitive Rous sarcoma virus decreases with cell density, but is found not significantly different at permissive and at non-permissive temperature. Kinetic analysis of 45Ca efflux from preloaded cells exhibits three separable pools of exchangeable calcium. The ratio of pool size of the fast-exchanging Ca-compartment (bound to cell surface) to pool size of the intermediate Ca-compartment (cytoplasmic) was found to decrease from 2.5 to 1.3 upon shift from non-permissive to permissive temperature. The slowly exchanging Ca-pool (presumably mitochondrial) did not change significantly upon temperature shift. These and further data demonstrate a close correlation between distribution of cellular Ca among different cellular compartments and characteristics of cellular proliferation, both attributable to the function(s) of a single oncogene.     

Kirsten murine sarcoma virus transformed cell lines and a spontaneously transformed rat cell-line produce transforming factors

We have examined culture fluids from a variety of Kirsten murine sarcoma virus (KiMSV) transformed rat and mouse cells for the presence of factors which induce normal Rat-1 cells to assume the transformed phenotype. All KiMSV transformants produced transforming factor (TF). Revertants of KiMSV transformed rat or mouse cells failed to relase TF as did normal rat or mouse cells. Cells transformed by a temperature sensitive mutant of KiMSV produced TF at the permissive temperature but not at the nonpermissive temperature. Further, cells from a spontaneous transformant of Rat-1 cells also produced TF. TF is a small polypeptide which competes for the epidermal growth factor receptor. Its effect upon normal cells is reversible and requires de novo RNA and protein synthesis. Cells treated with TF lose the actin fibers observed in normal fibroblasts, assume a transformed cell morphology, become anchorage independent for growth, grow in low concentrations of serum, grow to a high cell density, and have an increased rate of hexose uptake.     

Phosphorylations of the subcellular matrix in cells transformed by Rous' sarcoma virus

The transforming protein of Rous' sarcoma virus (RSV) is a phosphoprotein of Mr 60 000 (pp60src) which displays protein kinase activity specific for tyrosine residues; pp60src is associated with the plasma membrane and is recovered in the detergent-insoluble material which represents the subcellular matrix of the cell. After phosphorylation of this material of RSV-transformed cells with [gamma-32P]ATP, five phosphoproteins have been detected which are not seen in normal cells. These proteins (Mr = 135 000, 125 000, 75 000, 70 000, 60 000) contain phosphotyrosine. Their phosphorylation is strongly inhibited by anti-pp60src antibodies. In cells transformed by a temperature-sensitive mutant of RSV, these phosphoproteins, present at the permissive temperature, are no longer detected at the non-permissive temperature. It is concluded that these phosphorylations are mediated by pp60src protein kinase activity. This supports a possible role of the phosphorylation of cytoskeletal proteins in the transformation process.     

The role of calmodulin in cell transformation

Two cell lines transformed with temperature sensitive retroviruses were examined for: their ability to grow in low Ca²⁺ medium, their calmodulin levels and changes in calmodulin acceptor proteins. Both cell lines grow in low Ca²⁺ medium at the permissive temperature 34°C while both lines did not replicate at the non-permissive temperature 39°C. The NRKLA23 cells have nearly twice as much calmodulin at the permissive temperature than they do at the non-permissive temperature while the 6M2 cells have an equal amount of calmodulin at both temperatures. Both cell lines exhibit changes in the calmodulin acceptor proteins going from the permissive to the non-permissive temperature. We suspect that the changes in the calmodulin acceptor proteins may be involved in the altered Ca²⁺-sensitivity of growth in the cells going from the permissive to non-permissive temperature.     

Regulation of the proliferative response in Rous sarcoma virus transformed chicken embryo fibroblasts by serum and multiplication-stimulating activity (MSA).

A temperature sensitive mutant of Rous sarcoma virus (tsNY68) was used to obtain cultures of quiescent virus-infected chicken embryo fibroblasts arrested by serum starvation at the non-permissive temperature. Upon shift to the permissive temperature, these cells enter the replicative cell cycle as evidenced by increases in 2-deoxyglucose uptake, 3H-thymidine incorporation and percent labeled nuclei. These changes occur in the absence of serum and the cells become morphologically transformed within eight to ten hours after the temperature shift. Entry into the S phase temporally resembles that of normal quiescent fibroblasts stimulated with serum. This experimental system was used to examine the proliferative response of transformed cells to serum and purified multiplication-stimulating activity (MSA) during the transition from the resting to the growing state. Data are presented which show that the presence of serum in the medium enhances the proliferative response of quiescent infected cells shifted to the permissive temperature over those shifted in the absence of serum. In contrast, the presence of MSA has no additional effect on the response exhibited by infected cells shifted to the permissive temperature in serum-free medium. Labeled MSA binding experiments show that this lack of response is not due to a loss of MSA receptors on the cell surface since transformed cells are still capable of binding MSA at the same level as normal cells. The results are consistent with the hypothesis that the set of biochemical events initiated by MSA in normal cells are turned on in infected cells shifted to the permissive temperature by the activation of the src gene product.     

Elevated phosphatidylinositol kinase activity in Rous sarcoma virus-transformed cells. Lack of evidence for enzyme translocation

Phosphatidylinositol kinase (E.C. 2.7.1.67) activity of rat fibroblasts transformed by Rous sarcoma virus (RSV) was measured and compared with immunoprecipitated protein tyrosine kinase activity associated with pp60v-src. Both enzyme activities were elevated in the particulate fractions from wild-type RSV-transformed cells and cells transformed by a temperature-sensitive mutant of RSV when grown at the permissive temperature. The presence of the non-ionic detergent Nonidet P-40 in the phosphatidylinositol kinase assays stimulated the soluble and particulate forms of the enzyme to different degrees but did not affect the relative differences between transformed and untransformed cells. Our results indicate that phosphatidylinositol kinase activity is a good correlate of RSV transformation and suggest a functional relationship between pp60v-src and phosphatidylinositol kinase.     

Colloidal iron hydroxide-binding to the surfaces of chick embryo fibroblasts transformed by wild-type and a temperature-sensitive mutant of Rous sarcoma virus.

The densities of colloidal iron hydroxide (CIH) particles binding to the surfaces of chick embryo fibroblasts were determined before and after transformation with wild type Rous sarcoma virus and a temperature sensitive (ts) mutant of this virus. On the basis of in vitro behavior, cells transformed by the ts virus manifest a malignant phenotype at 36 degrees C (permissive temperature) and appear normal at 41 degrees C (non-permissive temperature). At the permissive temperatures there is a significant increase in CIH particle-binding to spaces of cell surface between microvilli on the wild type and ts transformed cells. At the non-permissive temperature this significant increase in binding is only observed on the wild type transformant, while the density found on the ts transformant is not significantly different from the untransformed state. Therefore, in vitro characteristics of normalcy and malignancy are reflected in changes in the CIH binding properties of the cell surface spaces between microvilli. The CIH densities observed on the microvilli are significantly different from the density on the spaces between them for each of the classes of cells studied at either temperature. The microvilli are found to bind a lower density of particles in five of the six cases. No correlations between microvilli particle density and transformation to in vitro malignant characteristics were observed.     

State of the viral DNA in rat cells transformed by polyma virus. II. Identification of the cells containing nonintegrated viral DNA and the effect of viral mutations.

F2408 rat cells transformed by polyoma virus contained integrated and nonintegrated viral DNA. The presence of nonintegrated viral DNA is under control of the A early viral function. Polyoma ts-a-transformed rat cells lose the free viral DNA when growth at the nonpermissive temperature (40 degrees C), but they reexpress it 1 to 3 days after they are shifted back to the permissive temperature. In contrast, rat cells transformed by a late viral mutant, ts-8, contain free viral DNA at both permissive and nonpermissive temperatures. Treatment of the transformed rat cells with mitomycin C produces a large increase in the quantity of free viral DNA and some production of infectious virus. Experiments of in situ hybridization, with 3H-labeled polyoma complementary RNA as a probe, show that only a minority (approximately 0.1%) of the transformed cells contain nonintegrated viral DNA at any given time. These results suggest that the presence of free viral DNA in polyoma-transformed rat cells is caused by a spontaneous induction of viral DNA replication, occurring with low but constant probability in the transformed cell population, and that the free viral DNA molecules originate from the integrated ones, probably through a phenomenon of excision and limited replication.     

Function of simian virus 40 gene A in transforming infection.

In productive infection by simian virus 40, the A gene is known to regulate the initiation of viral DNA replication and to control the synthesis of late viral RNA. The function of the A gene in transforming infection was investigated by the infection of a variety of cell species with six independently isolated temperature-sensitive mutants belonging to the A complementation group. The A mutants failed to initiate the stable transformation of cells during continuous infection at the restrictive temperature. After the establishment of transformation at the permissive temperature and a subsequent shift to the restrictive temperature to block the A function, however, two distinct virus-cell interactions were identified. In one case, the increased colony-forming capacity of transformed cells remained stable after the temperature shift. In the other case, the temperature shift decreased the capacity of transformed cells to form colonies to the level of untransformed control cells. The outcome of the virus-cell interaction depended both on the nature of the A mutation in a given cell species and on the species of the cell transformed by a given mutant. These findings suggest that the transformation process may require two distinct events, each related to A gene expression.     

Studies on the synthesis and structure of mitochondrial DNA in cells infected by rous sarcoma viruses and on the occurrence of intramitochondrial virus-like particles in certain RSV-induced tumor cells

1. The synthesis of mitochondrial DNA in CEF in vivo at 3,4 and 6 days after infection with RSV (Schmidt-Ruppin, subgroup A) was progressively stimulated 2 to 4-fold as compared with that in uninfected CEF cells grown in parallel. 2. The stimulation of mtDNA synthesis in vivo upon transformation was found to be temperature dependent when the thermosensitive mutant of RSV, T5, was used to infect the cells. 3. In contrast to mtDNA synthesis, nuclear DNA synthesis did not differ in transformed and uninfected cells, nor did it change significantly upon temperature shifts. 4. MtDNA (monomeric and catenated dimeric forms) in transformed and uninfected CEF replicate by displacement synthesis. Various replication intermediates are described. 5. The restriction endonuclease EcoRI cleaves closed circular mtDNA from CEF at one specific site. 6. Heteroduplex molecules formed between nicked circular and/or EcoRI cleaved mt DNA molecules from uninfected and transformed CEF revealed, with a few exceptions, no detectable base sequence heterogeneity in at least 98% of cases. 7. Intramitochondrial virus like particles (IMV) are described in hamster tumor cells. The evidence suggests both engulfment of cytoplasmic particles by mitochondria and the presence of intramitochondrial incomplete forms of particles. Bromodeoxyuridine was found to enhance the frequency of IMV.     

Properties of permissive monkey cells transformed by UV-irradiated simian virus 40.

African green monkey cells (CV1 line) were infected with UV-irradiated simian virus 40 (SV40), and permissive lines of stably transformed cells were established. These cell lines display the SV40 T-antigen and the growth characteristics typical of nonpermissive transformed cells (e.g., reduced cell density inhibition, reduced serum dependence, ability to overgrow normal cells, and colony formation in soft agar). The level of permissiveness to superinfecting SV40 is fully comparable with that of nontransformed CV1 and BSC-1 lines. The transformed monkey lines also support SV40 plaque production under agar. By Cot analysis, the transformed permissive cells contain, on an average, 1 to 2 SV40 genome equivalents, and the majority of the viral sequences are associated with the high-molecular-weight cellular DNA. No spontaneous production of infectious SV40 has been observed. The transformed permissive monkey cells failed to support the replication of SV40 tsA mutants at the restrictive temperature. To account for this, it is suggested that the gene A product has separate functions for transformation and initiation of viral DNA synthesis, and only the former function is expressed in the transformed permissive monkey cells.     

Role of cell division in differentiation of myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus.

The relationship between a potential requirement for cell DNA synthesis and the expression of differentiated muscle cell functions was investigated using primary chicken embryo myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus (RSV). Under optimized conditions, transformed myoblasts growing at the permissive temperature could differentiate into multinucleated myotubes, express muscle-specific myosin, desmin and acetylcholine receptors in the absence of DNA synthesis and cell division following a shift to the non-permissive temperature. Furthermore, the experiments demonstrate that individual RSV-infected myoblasts have two options: either to divide and express the transformed phenotype or to withdraw from the cell cycle and differentiate into myotubes. The choice between these options appears to depend on the protein-kinase activity of pp60src, the src gene product.     

Initiation of DNA replication in Escherichia coli

Summary When E. coli F⁺ cells carrying the dna-167 or dnaC2 mutation, which causes the temperature-sensitive initiation of DNA replication, are exposed to a non-permissive temperature to stop the replication of chromosome and F factor, and then transferred back to a permissive temperature with the addition of chloramphenicol, one round of the chromosomal replication occurs, but further replication is inhibited. Under these conditions, F DNA replicates coincidentally with the initiation of the chromosomal replication in both strains. When rifampicin is added to the cells upon lowering of the temperature, the chromosome can not replicate in the F⁺ dna-167 strain, but can do so in the F⁺ dnaC2 strain. F DNA can replicate in both of the mutant strains under these conditions.     

Regulation or procollagen messenger ribonucleic acid levels in Rous sarcoma virus transformed chick embryo fibroblasts

Using cloned cDNAs for pro-alpha 1 and pro-alpha 2 collagen messenger ribonucleic acid (mRNA), we have investigated the regulation of collagen mRNA levels in Rous sarcoma virus (RSV) transformed chick embryo fibroblasts (CEF). We find that both pro-alpha 1 and pro-alpha 2 mRNA levels are decreased approximately 10-fold in CEF transformed by either the Bryan high-titer strain or the Schmidt-Ruppin strain of RSV. Using temperature-sensitive mutants in the transforming gene src, we also investigated the rate of change in the levels of the two mRNA species. We employed mutants of both the Bryan high-titre strain (BHTa) and the Schmidt-Ruppin strain (ts68). With both mutants the results were similar. Upon shift from the permissive temperature (35 degrees C) to the non-permissive temperature (41 degrees C), collagen mRNA synthesis, did not increase until more than 5 h had passed, suggesting that action of src on collagen gene expression is indirect. Upon shift from 41 to 35 degrees C, collagen mRNA levels fell with a half-life of 10 h. Whether this fall reflects the half-life of procollagen mRNA or an effect of src on procollagen RNA stability is unclear. Both pro-alpha 1 and pro-alpha 2 mRNA levels were coordinately controlled.     

The effects of an Escherichia coli dnaAts mutation on the replication of the plasmids colE1 pSC101, R100.1 and RTF-TC.

Summary The rate of replication of the plasmids colE1, pSC101, R100.1 and pAR132 (an RTF-TC derivative of the drug resistance factor R100.1) has been investigated directly by DNA: DNA hybridization. These rates have been compared, in a dnaAts strain, to that of various markers of the host chromosome at permissive and non-permissive temperatures. Chromosome initiation in the dnaAts strain stops rapidly after a shift to the non-permissive temperature, but plasmids R100.1 and pAR132 do not seem to be affected directly and continue replication for some time. The colE1 replication rate undergoes a large increase after the temperature shift, followed by a rapid decrease to a very low level 25 min after the shift. In contrast pSC101 replication stops immediately after the shift. ColE1 is able to replicate in an integratively suppressed dnaAts strain at 42° C whereas pSC101 stops replication immediately under these conditions. We conclude that R100.1 and its derivative RTF-TC can replicate without a functional dnaA product; that colE1, while affected by a shift in temperature in a dnaAts strain, does not directly require dnaA; and that the plasmid pSC101 has an absolute requirement for dnaA. The absolute requirement of pSC101 for dnaA in the integratively suppressed Hfr strain provides a useful system for further investigations of the dnaA function.     

Tumor progression mediated by two cooperating DNA segments of human cytomegalovirus.

The terminal fragments (EJ and EM) of the XbaI-E transforming segment of human cytomegalovirus can independently induce the tumorigenic conversion of immortalized cells. To study their interaction, Rat-2 cells were transfected singly or with a combination of cloned EJ and EM DNAs. Large transformed foci were induced at a 10-fold higher frequency by EJ plus EM than by either DNA fragment alone. Focus-derived lines transformed by EJ plus EM produced tumors in syngeneic rats at a much faster rate (5 to 7 days) than did cell lines transformed by EJ or EM alone (25 to 35 days). Southern hybridizations showed that EM-homologous DNA was retained, exhibiting a complex pattern of multiple and amplified bands in EJ-plus-EM lines compared to a simple pattern in EM-induced lines. EJ DNA was not detected in the single or double transformants. The levels of p29, a 29-kilodalton transformation-sensitive marker in Rat-2 cells, were decreased 10- to 100-fold in cell lines transformed by EJ or EM fragment alone. Synthesis of p29 was shut off in EJ- plus-EM transformants. These data demonstrate that two unlinked transforming regions of human cytomegalovirus can cooperate to produce an aggressive tumorigenic phenotype.     

Isolation and analysis of a mammalian temperature-sensitive mutant defective in G2 functions

A temperature-sensitive (ts) mutant, designated tsFT210, was isolated from a mouse mammary carcinoma cell line, FM3A. The tsFT210 cells grew normally at 33 degrees C (permissive temperature), but more than 80% of the cells were arrested at the G2 phase at 39 degrees C (non-permissive temperature) as revealed by flow-microfluorimetric analysis. DNA replication and synthesis of other macromolecules by this mutant seemed to be normal at 39 degrees C for at least 10 h. However, in this mutant, hyperphosphorylation of H1 histone from the G2 to M phase, which occurs in the normal cell cycle, could not be detected at the non-permissive temperature. This suggests that a gene product which is temperature-sensitive in tsFT210 cells is necessary for hyperphosphorylation of H1 histone and that this gene product may be related to chromosome condensation.     

Early changes in the synthesis of proteins with affinity for single-stranded DNA during the onset of transformation in NRK cells.

Early alterations in the synthesis of proteins which bind to single-stranded DNA have been examined following the onset of transformation in NRK cells transformed by a heat-sensitive mutant (ts339) of Rous sarcoma virus. Transformation was initiated by shifting quiescent cultures from nonpermissive to permissive temperatures. Cultures were prelabelled with [3H]leucine for several generations at the non-permissive temperature, and with [35S]methionine at times after shift to the permissive temperature. Cytosol extracts were passed through sequential columns of double-stranded and single-stranded DNA bound to cellulose. Within the first hour of transformation there was an increase in the synthetic rate of proteins binding tightly to single-stranded DNA, but not to double-stranded DNA. More loosely bound protein fractions showed no such early synthetic increase. Electrophoresis of the fraction eluted from single stranded DNA-cellulose with 2 M NaCl demonstrated the presence of a major protein of 93 000 daltons, which comprised more than 0.1% of the cytosol protein. The synthesis of the 93 000 dalton protein increased continuously over the first 4 h interval after the onset of transformation. The synthetic rate of a 35 000 dalton protein, a major DNA-binding polypeptide found in mammalian cells, began to increase after a 1-h lag, following the onset of transformation. The protein fraction containing the 93 000 dalton protein had considerable unwinding activity, depressing the melting temperature of poly(dA-dT) by 39 degrees C. The protein fraction containing the bulk of the 35 000 dalton protein did not have unwinding activity. Transformation-induced DNA synthesis was measured in cells made permeable to deoxyribonucleoside triphosphates at times after shift to the permissive temperature. It was determined that synthesis of DNA began within the first 1--2 h after the onset of transformation. We conclude that the early transformation-associated synthesis of SS93 and perhaps other proteins binding to single-stranded DNA may be related to early transformation-associated changes preparatory to DNA replication and subsequent growth.