Growth control of normal and transformed cells

Both serum factors and protein synthesis are required for normal cell growth. Swiss 3T3 cells require the serum growth factors insulin and EGF (epidermal growth factor) during the initial part of the G₁ period, until they pass a restriction point about 2 h before the initiation of DNA synthesis. Concentration of cycloheximide that inhibit protein synthesis by as much as 70% dramatically lengthen the cell cycle before the restriction point, while the cell cycle after the restriction point remains nearly constant. These results are consistent with a model in which labile proteins are required for transit of cells past the serum-sensitive restriction point. The relation of these findings to the growth control of transformed cells is discussed.     

Characterization of a serum-free culture system comparing growth factor requirements of transformed and untransformed cells

We describe the first completely serum-free model culture system for comparing growth control in transformed and untransformed cells. Continuous maintenance of untransformed AKR-2B fibroblasts and chemically transformed AKR-MCA cells in the presence of serum-free medium containing epidermal growth factor (E), insulin (I), and transferrin (T) resulted in cell lines which proliferated with similar doubling times (14 h), comparable to parental lines maintained in 10% serum (16 h). The transformed MCA-SF cells and untransformed AKR-SF cells did not differ in their saturation densities in medium containing E + I + T. However, the monolayer proliferation of MCA-SF cells was significantly greater than that of the AKR-SF cells in the presence of E + T, I + T, or T alone. Both cell lines required T to proliferate in monolayer culture. [3H]-Thymidine incorporation experiments and autoradiographic analysis indicated that quiescent MCA-SF cells could reenter the cell cycle by addition of nutrients alone. The combination of E + I + T produced no additional stimulation of DNA synthesis. In contrast, individual polypeptide growth factors (E, I, IGF-I, PDGF, FGF a or b, or TGF-beta 1) were required to elicit a mitogenic response in the untransformed AKR-SF cells. Peak mitogenesis occurred from 18-20 h for all growth factors except TGF-beta 1 (32 h). Neither AKR-SF nor MCA-SF cells could grow with anchorage independence in serum-free medium, unless both TGF-beta 1 and FGF a or b were simultaneously present. The results indicate that this well-defined, serum-free model system can be utilized to detect growth factor-related alterations associated with the transformed state.     

Serum-free growth of normal and transformed fibroblasts in milk: differential requirements for fibronectin

Bovine milk may be used as a supplement for the serum-free growth of certain fibroblastic cells in culture. The growth properties of three representative cell types in milk-supplemented medium were examined; fibroblastic cell strains, fibroblastic cell lines, and transformed fibroblasts. Transformed fibroblasts, which included RNA and DNA tumor virus-transformed cells and carcinogen-transformed cells, grew in milk. Instead of growing attached to the culture dishes, as they normally do in serum, transformed fibroblasts grew in milk as large clusters in suspension. In contrast, nontransformed fibroblastic cell strains and cell lines did not grow in milk-supplemented medium. Fibroblasts transformed by a temperature-sensitive transformation mutant of Rous sarcoma virus were temperature-sensitive for growth in milk. The failure of cells to adhere to the substratum in milk-supplemented medium suggested that milk might be deficient in attachment factors for fibroblasts. When the attachment of fibroblastic cells in milk- supplemented medium was facilitated by pretreating culture dishes with fibronectin, (a) transformed cells grew attached rather than in suspension, (b) normal cell lines attached and grew to confluence, and (c) normal cell strains adhered and survived but did not exhibit appreciable cell proliferation.     

Differential in vitro growth properties of cells transformed by DNA and RNA tumor viruses

Mammalian cells transformed by DNA and RNA tumor viruses are shown to display consistently different growth properties. All SV40, adenovirus type 7 and polyoma virus (DNA viruses) transformed cells propagated to high densities. The same cells transformed instead by RNA viruses: MSV strain Kirsten (MSV-Ki) or MSV strain Maloney (MSV-M) grew to densities which were consistently lower than DNA virus-transformed cells but greater than that of untransformed cells. The capacity to synthesize DNA at increasing densities also differentiated the RNA and DNA virus-transformed cells. As growing cultures of untransformed cells neared saturation density, the fraction of cells synthesizing DNA was minimal. The RNA virus-transformed cells were also contact-inhibited but at a significantly higher density. In contrast the DNA virus-transformed cells propagated to still greater densities and continued DNA synthesis at a high rate even at very high densities. Therefore the DNA virus-transformed cells truly are not contact inhibited. It is suggested that the capacity to continue DNA synthesis at high densities explains the attainment of much greater densities by DNA virus-transformed cells. There were no clear-cut differences in the ability to form colonies in agar, although a few of the RNA virus-transformed lines could not be propagated in semi-solid medium. These results may be explained as a persistence of the capacity of DNA tumor viruses to stimulate host cell DNA synthesis.     

The regulation of DNA repair processes in mammalian cells. II. The repair of DNA radiation damage in NIH 3T3 murine cells transformed by the v-myc oncogene]

The kinetics of repair of the ionizing radiation-induced DNA single- and double-strand breaks in the normal NIH 3T3 mouse cells and in those transformed with virus oncogenes v-myc has been investigated. The incubation of non-transformed cells for 18 hours in serum-free medium results in significant decrease in the rate of the single-strand DNA breaks repair during the first minutes of post-irradiation incubation. This effect is absent in transformed cells. The DNA double-strand breaks repair is more efficient in transformed NIH 3T3 cells as compared to that in the non-transformed ones both after their incubation in the medium with 10% fetal bovine serum or without serum. However, more significant differences in the rate of elimination of these DNA lesions was found in the serum-free medium. Hence, the presence of v-myc sequences in the transformed cells prevented from a decrease in the efficiency of DNA repair due to incubation of cell culture in serum-free medium. These results agree with the assumption that c-myc gene product may be a mediator in regulation of DNA repair by the epidermal growth factor. These data also show that the c-myc gene expression in an important condition providing a high efficiency of the constitutive DNA repair process.     

Role of the simian virus 40 gene A product in regulation of DNA synthesis in transformed cells.

Cells transformed by tsA mutants of simian virus 40 (SV40) are temperature sensitive for the maintenance of the transformed phenotype. The kinetics of induction of DNA synthesis were determined for hamster cell transformants shifted to the permissive temperature after a 48-h serum arrest at the nonpermissive temperature. DNAsynthesis was initiated in the tsA transformants by 8 h after shiftdown was maximal by 12 h. The presence or absence of fetal bovine serum at the time of temperature shift had no effect on the kinetics of initiation of DNA synthesis. Analysis of TTP in tsA transformants revealed similar levels of incorporation of [3H]thymidine into TTP at both permissive and nonpermissive temperatures. Autoradiography revealed that by 12 h after a shift to the permissive temperature, approximately 50% of the cells exhibited labeled nuclei after a 60-min pulse with [3H]thymidine, indicating that a majority of the cells were actively synthesizing DNA. By 8 to 12 h after a shiftup of confluent tsA transformants to the nonpermissive temperature, the number of labeled nuclei was reduced to approximately 16%, regardless of serum concentration. These data indicate that the SV40 gene A product, either directly or indirectly, regulates cellular DNA synthesis in transformed cells.     

Involvement of protease in L-glutamine control of nucleic acid and polyphosphate metabolism in cells transformed by 9,10-dimethyl-1,2-benzanthracene, SV40 and H-ras oncogene

Mammalian cells transformed with either 9,10-dimethyl-1,2-benzanthracene, SV40 or H-ras oncogene dramatically changed their ability to synthesize DNA and RNA and metabolize polyphosphate when L-glutamine was withdrawn from the growth medium or when heat shocked (growth at 42 degrees C). Untransformed, DNA and RNA synthesis decreased by 50-80% when glutamine was withdrawn, but polyphosphate accumulated whether or not glutamine was supplied. Heat shock did not alter this response. Transformed isogenic cells responded differently; at 37 degrees C, they decreased their synthesis of DNA and RNA if starved for glutamine, whereas at 42 degrees C, synthesis was optimal without glutamine. Transformed cells accumulated polyphosphate at 37 degrees C when starved for glutamine, but at 42 degrees C, no polyphosphate accumulated. This apparent non-dependence on glutamine by transformed cells when heat shocked was found to be due to the production of glutamine from serum proteins through induction of a protease(s).     

Regulation of polyamine biosynthesis in normal and transformed hamster cells in culture

Several aspects of polyamine biosynthesis were compared in low-passage hamster embryo fibroblasts and transformed hamster fibroblasts. Earlier studies had demonstrated a larger and longer-lasting induction of ornithine decarboxylase activity in transformed cells than in hamster embryo fibroblasts. The increases in intracellular polyamine concentrations after serum stimulation were much greater in chemically transformed HE68BP cells than in normal hamster fibroblasts. Treatment of confluent cultures with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, greatly potentiated ornithine decarboxylase induction by fresh medium in HE68BP cells, but not in hamster fibroblasts. A similar synergistic effect was observed when transformed cells, but not normal cells, were treated with the combination of insulin and promoter. HE68BP cells were capable of growth in medium containing serum concentrations as low as 0.5%, whereas only concentrations of 5% or more supported the growth of hamster embryo fibroblasts. Low serum concentrations induced ornithine decarboxylase in HE68BP cells but not in normal cells, and a given serum concentration always produced a greater induction of ornithine decarboxylase in transformed than in normal cells.Another enzyme involved in polyamine synthesis, $\text{S-}\text{adenosyl-}\text{L}\text{-methionine}$ decarboxylase was induced in normal and transformed cells by serum-containing medium or tetradecanoylphorbol acetate, but in contrast to ornithine decarboxylase, no synergistic effect was seen in transformed cells exposed to the combination of fresh medium and the tumor promoter. A macromolecular inhibitor of ornithine decarboxylase was readily detected in hamster fibroblast cultures treated with high concentrations of putrescine, but little or none of this inhibitor was found in HE68BP cultures. In both cell types, however, serum induction of ornithine decarboxylase was inhibited under conditions of excess putrescine.The results demonstrate several differences between normal and transformed hamster cells in the regulation of polyamine synthesis.     

Differences between diploid and transformed human cells in the incorporation of 5-bromodeoxyuridine into DNA

In diploid human cells, the DNA precursor pool equilibration times for exogenous thymidine are about twice those for the thymidine analogue 5-bromodeoxyuridine (BUdR); in cells that were either transformed chemically or derived from malignant tumours, the pool equilibration times are the same for thymidine and 5-bromodeoxyuridine and are closer in value to the shorter (bromodeoxyuridine) times of the diploid cells. Thymidine, if present in the culture medium with BUdR, is incorporated into DNA preferentially in diploid cells (by 2 or 3 to 1). Discrimination against bromodeoxyuridine is evident within 2 h of incubation of the two precursors with diploid cells, but is not observed even after 24 h in any of the transformed cell lines tested. Experiments were performed to test the effect of inhibitors of the mammalian DNA polymerases alpha (N-ethylmaleimide) and beta (incubation of cells at 45 °C) upon the ability of cells to synthesise DNA and to incorporate thymidine preferentially when present with equimolar BUdR. In diploid cells, overall in vivo DNA synthesis is more sensitive to N-ethylmaleimide and more resistant to 45 °C treatment than is DNA synthesis in the transformed cell lines. N-Ethylmaleimide decreases the capacity of diploid cells to discriminate against BUdR, whereas heating increases it. Transformed cells treated with N-ethylmaleimide remain unable to discriminate against BUdR; some transformed lines, when heated at 45 °C, become less incapable of such discrimination.     

Effects of serum and conditioned medium on protein degradation, migration of nonhistone proteins to the nucleus, and DNA synthesis in transformed cells

Stimulation of resting transformed cells (Chang liver cells), prelabeled with [3H] leucine, with fetal calf serum, caused increased nuclear translocation of [3H] nonhistone proteins ([3H] NHP) and DNA synthesis and a parallel inhibition of proteolysis of cellular proteins. [3H] NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5-6.5, showed that [3H] NHP fractions with high degradation rates in resting cells corresponded to the [3H] NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H] NHP are linked. Conditioned medium (COM) produced by Chang cells had similar effects as serum, suggesting that factors produced by these transformed cells, control cell growth by a mechanism that is similar to serum. The lysosomotropic amine eserine had similar effects as serum and COM. Based on the similarity of the effects, it would appear that serum and COM inhibit lysosomal proteolysis. It is proposed that serum and COM induce NHP migration to the nucleus by inhibiting lysosomal degradation of these proteins. Serum and COM caused also migration of [3H] histones to the nucleus, however the mechanism is not clear.     

Loss of density-dependent regulation of multiplication of BALB-3T3 cells chemically transformed in vitro

Balb/3T3 cells show density-dependent regulation of multiplication with the final cell density depending on serum concentration in the media. Chemically transformed Balb/3T3 cells (Balb/3T3-D) pile up on each other, multiply to a high cell density, but have decreased DNA synthesis at very high cell densities. Balb/3T3-D cells require less serum for multiplication compared with original Balb/3T3 cells. A rat serum fraction and a bovine β-globulin fraction stimulate the multiplication of Balb/3T3 cells but only slightly stimulate Balb/3T3-D cells indicating different serum factors stimulate growth of these two cell types. The multiplication properties of Balb/3T3-D cells are very similar to those of SV-40 transformed 3T3 cells, however, these properties were brought about by a single treatment by a chemical carcinogen, without an exogenous virus. The transformation altered the contact of cells to one another, indicating a permanent chemical change in the membrane structure.     

Regulation of DNA replication by serum and the transforming function in cultured rat fibroblasts transformed by Rous sarcoma virus.

The onset and rate of semiconservative DNA replication were measured in stimulated cultured rat fibroblasts and their Rous sarcoma virus-transformed derivatives after a period of serum deprivation. Rat-1 (tsLA24/RSV) cells initiated DNA synthesis following a shift to the permissive temperature or addition of serum at the non-permissive temperature. Their rate of DNA replication was unaffected by the presence of serum at the permissive temperature, however, there was a serum requirement at the non-permissive temperature. The transition probability was less at the permissive temperature, independent of serum, than at the non-permissive temperature in the presence of serum. The amount of DNA induced to replicate by addition of serum at the non-permissive temperature or by a shift to the permissive temperature was similar. Using the untransformed Rat-1 cells and these cells transformed by wild-type RSV (Rat-1 (wt/RSV)), it was confirmed that the rate of entry into S phase (transition probability) was always lower in the transformed cell line at both 39° and 35°. In both cell lines the rate of DNA replication was independent of temperature, but the onset was delayed at the lower temperature. These results indicate that in the cell lines examined, (1) serum was able to commit the cells to replicate DNA (alter the transition probability) in both transformed and untransformed cells, but the transforming function was able to supplant a serum-dependent process during G₁ necessary for the initiation of DNA replication, and (2) the effects of the transforming function and serum factor(s) on the alteration of the transition probability are not additive, suggesting that the transforming function initiates a process which acts at the level of the commitment to DNA replication which may render the normal serum-related control mechanisms ineffective in the regulation of growth.     

Virogenic Properties of Bromodeoxyuridine-sensitive and Bromodeoxyuridine-resistant Simian Virus 40-transformed Mouse Kidney Cells

When simian virus 40 (SV40)-transformed mouse kidney cells (mKS) were grown in the presence of susceptible indicator cells, SV40 was readily recovered from: (i) 15 transformed cell lines, (ii) transformed cells subcultured 45 times over a 7-month period in medium containing antiviral serum and bromodeoxyuridine (dBU), (iii) 45 of 46 clonal lines isolated in the presence of antiviral serum, (iv) 19 of 19 secondary clones isolated from two clonal lines, and (v) dBU-resistant transformed cell lines. dBU-resistant SV40-transformed mouse kidney cell lines were selected and shown to contain the T antigen and to have normal levels of thymidylate kinase and deoxyribonucleic acid (DNA) polymerase, but to be deficient in thymidine (dT) kinase. Radioautographic and biochemical experiments demonstrated that very little (3)H-dT was incorporated into DNA of dBU-resistant cells during a 6-hr labeling period. After infection of dT kinase-deficient mKS cells with vaccinia virus, high levels of dT kinase were induced. The properties of SV40 recovered from dBU-sensitive and dBU-resistant cells were studied. SV40 recovered from transformed cells was shown to express in CV-1 cells at least six functions characteristic of parental virus: synthesis of capsid antigen, synthesis of T antigen, synthesis of viral DNA, induction of dT kinase, induction of DNA polymerase, and induction of host cell DNA synthesis. In addition, SV40 recovered from the transformed cells induced T antigen, dT kinase, deoxycytidylate deaminase, thymidylate kinase, and DNA polymerase in abortively infected mouse kidney cultures, and the virus was also capable of transforming primary cultures of mouse kidney cells.     

Changes in the uptake of 2-deoxy-D-glucose in BALB-3T3 cells chemically transformed in culture

Balb/3T3 cells transformed in culture by chemical carcinogens were shown to multiply in a medium supplemented with 2% calf serum or with 10% agamma new-born calf serum. The cell lines that multiply well in medium supplemented with 10% agamma serum produced a higher incidence of tumors in X-irradiated weanling mice than the lines that multiply poorly. The difference in 2-deoxy-D-glucose uptake into exponentially growing transformed and un-transformed cells was 50–100%. In crowded cultures untransformed Balb/3T3 cells ceased taking up the sugar, while chemically transformed cells continued at the same rate even at high cell densities; thus, the difference became greater in crowded cultures. When the serum concentration in the media was reduced from 10% to 2%, untransformed Balb/3T3 cells took up the sugar at a reduced rate, while chemically transformed cells were only slightly affected; agamma new born calf serum supplemented medium had no effect on sugar uptake in any of the cells. When the serum concentration was changed from 2% to 10%, untransformed cells increased sugar uptake followed by cell division. The immediacy (within 15 min) of the response in the sugar uptake to 10% serum concentration suggested that the increased uptake rate and the consequent higher concentration of the sugar (D-glucose in normal situation) within Balb/3T3 cells triggered the cell cycle. Chemical carcinogens appear to alter permanently the uptake mechanism for a key nutrient.     

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.     

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.     

Hyaluronate synthetase inhibition by normal and transformed human fibroblasts during growth reduction

To establish the relation of glycosaminoglycan synthesis to cell proliferation, we investigated the synthesis of individual glycosaminoglycan species by intact cells and in a cell-free system, using normal and transformed human fibroblasts under differing culture conditions. Reducing serum concentration brought about a marked decline in the synthesis of hyaluronate (HA) as well as cell proliferation on both normal and transformed cells. Both HA synthesis and proliferation decreased with increasing cell densities markedly (in inverse proportion to cell density) in normal cells but gradually in transformed cells. This noticeable congruity of the changes in HA synthesis and proliferation indicates that the change in HA synthesis is related primarily to cell proliferation rather than to cell density or cellular transformation. Examination of HA synthesis in a cell-free system demonstrated that the activity of HA synthetase also fluctuated in conjunction with cell proliferation. Furthermore, growth-reduced cells (except crowded transformed cells) inhibited cell-free HA synthesis and this inhibition was induced coincidentally with a decrease in both HA synthetase activity and proliferation. These findings suggest that the change in HA synthesis is significant in the regulation of cell proliferation.     

Differential sensitivity of normal and transformed human cells to reovirus infection.

Normal and simian virus 40-transformed WI-38 cells exhibited a differential sensitivity to infection with type 3 reovirus. A progressive decrease in viability began 24 to 36 h after infection of transformed cells terminating in complete lysis of cultures by 96 h. Normal cells were productively infected and continued to produce and release virus for as long as 14 days after infection, but exhibited no detectable cytopathology. Inhibition of cellular DNA synthesis began 15 to 18 h after infection in transformed cells before development of cytopathology. No inhibition of DNA synthesis was detected in infected normal cells. No significant differences were noted in the adsorption or early replication characteristics of reovirus in normal and transformed cells. Virus replication and host cell DNA synthesis in normal and transformed human cells were compared to reovirus-infected L-929 mouse fibroblast cells.     

Low tumorigenicity of canine cells transformed by the human cytomegalovirus

Dog embryo kidney cells transformed by the human cytomegalovirus (HCMV) were obtained after non-permissive infection or transfection with viral DNA digested by restriction endonuclease EcoR I. The transformed cells, growing rapidly and showing an unlimited division potential, could use medium with only 2% serum for growth, contained nuclear virus antigens, and formed small colonies (less than 0.2 mm) in agarose. From 40 mice inoculated with transformed canine cells, only one eventually developed a tumor. Results indicate that dog cells are immortalized but not tumorigenically transformed by the human cytomegalovirus.