Biosynthesis of plasma membrane components by SV40-virus-transformed 3T3 mouse cells temperature sensitive for expression of some transformed cell properties

We have studied the plasma membranes of an SV40-transformed 3T3 cell line temperature sensitive for the transformed growth phenotype (ts H6-15 cells), and have found that they vary little as a function of temperature of cultivation. Analysis by polyacrylamide gel electrophoresis was performed on plasma membranes prepared from ts H6-15 cell cultured at the permissive (32 °C) and non-permissive (39 °C) temperatures and radioactively-labelled in several ways. No significant differences were seen when the electrophoretic patterns of polypeptides of the plasma membranes of ts H6-15 cells, grown through 3–4 generations in medium containing radioactive leucine (32 °C and 39 °C temperatures) were compared. Plasma membranes derived from cells similarly grown in medium with radioactive glucosamine indicated that extensive alterations in the intrinsic glycopeptides occurred in association with alteration in growth phenotype. A shift towards decreased synthesis of large molecular weight (? 100 000–160 000) glycopeptides occurred in cells grown at the temperature of non-transformed growtn (39 °C). A decrease in amount of a 1200 000 molecular weight glycopeptide at 39 °C was the most prominent of these alterations.We have studied the surface exposure of polypeptides and glycopeptides of intact cells grown at 32 and 39 °C, using lactoperoxidase-catalyzed iodination, NaBH₄ reduction of galactose oxidase-treated cells, and metabolic-labelling with glucosamine of trypsin-sensitive molecules. We found no major qualitative differences between whole cell extracts or between plasma membrane preparations of cells cultivated at the permissive and non-permissive temperatures. Of special interest was the observation that the formation and surface exposure of a trypsin-sensitive, 240 000 molecular weight polypeptide appeared not to be ts in ts H6-15 cells. The significance of these observations will be discussed.     

A consideration of the role of cell surface macromolecules in the process of viral transformation.

There is extensive physiological evidence implicating the cell surface as the key organelle which mediates the cell:cell interactions which underlie both normal and neoplastic growth. This information has now been supplemented with biochemical and biophysical data which indicates that surface macromolecules, in particular the heteroglycans of transformed cells, differ from those which lie at the periphery of normal cells. In the case of cells neoplastically transformed by most tumour viruses it is clear that the small virus genome (2-5 x 10(6) daltons) cannot carry the total genetic information to accomodate these various biochemical modifications, if indeed they are encoded in separate genes (1). To examine the part played in transformation by cellular genes coding for surface heteroglycan formation, we have turned to a study of SV-3T3 cells (ts H6-15) which are temperature-sensitive for expression of the transformed cell phenotype (2). The data show that cells grown under conditions permissive and non-permissive for such expression exhibit the same pattern of formation of glycolipids, and the majority of the polypeptides of the plasma membrane. There are, however, significant differences in the synthesis of some glycopeptides. A large molecular weight, trypsin-labile glycopeptide, present at the surface of untransformed fibroblasts but barely measurable in some of their virus-transformed derivatives (3), was detected, essentially at the same level, at the surface of ts H6-15 cells grown at the permissive and non-permissive temperatures. The signficance of these observations is discussed.     

A consideration of the role of cell surface macromolecules in the process of viral transformation

Summary There is extensive physiological evidence implicating the cell surface as the key organelle which mediates the cell:cell interactions which underlie both normal and neoplastic growth. This information has now been supplemented with biochemical and biophysical data which indicates that surface macromolecules, in particular the heteroglycans of transformed cells, differ from those which lie at the periphery of normal cells. In the case of cells neoplastically transformed by most tumour viruses it is clear that the small virus genome (2–5×10⁶ daltons) cannot carry the total genetic information to accomodate these various biochemical modifications, if indeed they are encoded in separate genes (1). To examine the part played in transformation by cellular genes coding for surface heteroglycan formation, we have turned to a study of SV-3T3 cells (ts H6-15) which are temperature-sensitive for expression of the transformed cell phenotype (2). The data show that cells grown under conditions permissive and non-permissive for such expression exhibit the same pattern of formation of glycolipids, and of the majority of the polypeptides of the plasma membrane. There are, however, significant differences in the synthesis of some glycopeptides. A large molecular weight, trypsin-labile glycopeptide, present at the surface of untransformed fibroblasts but barely measurable in some of their virus-transformed derivatives (3), was detected, essentially at the same level, at the surface ofts H6-15 cells grown at the permissive and non-permissive temperatures. The significance of these observations is discussed. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Factors affecting steroid and prostaglandin secretion by reproductive tissues of cycling and pregnant sows in vitro

Mitochondrial, microsomal and pellicular membranes were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C, and phospholipids, in turn, were separated from total lipids extracted from these membranes. The effect of growth temperature on their solid-to-fluid phase transition temperature was examined by wide-angle X-ray diffraction. The transition temperatures of phospholipids from mitochondria, microsomes and pellicles were 21, 19 and 26 degrees C for cells grown at 39 degrees C and -8, -3 and 6 degrees C for cells grown at 15 degrees C, respectively. All phospholipids were found in a completely fluid state at these growth temperatures. From a comparison between the phospholipids and total lipids from pellicles of cells grown at 39 degrees C, a triterpenoid alcohol, tetrahymanol, caused the transition temperature to increase. The alignment of tetrahymanol in membranes was examined with pellicle'a total lipid oriented in a sample holder.     

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.     

Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus.

The synthesis and processing of virus-specific precursor polypeptides in NIH/3T3 cells infected at the permissive temperature (31 degrees C) with temperature-sensitive (ts) mutants of Rauscher murine leukemia virus was studied in pulse-chase experiments at the permissive and nonpermissive (39 degrees C) temperatures. The newly synthesized virus-specific polypeptides were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera against Rauscher murine leukemia virus proteins. In cells infected with ts mutants defective in early replication steps (the early mutants ts17 and ts29), and ts mutants defective in postintegration steps (the late mutants ts25 and ts26), the processing of the primary gag gene product was impaired at the nonpermissive temperature. gag-pr75 of all four mutants was converted into gag-pr65; however, gag-pr65 accumulated at the nonpermissive temperature, and the main internal virion polypeptide p30 was not formed. Therefore, the proteolytic cleavage is blocked beyond gag-pr65. Concomitantly, the formation of the env gene-related polypeptide p12(E) of all four mutants was blocked at the restrictive temperature. In contrast, cells infected with the late mutant ts28, which produced noninfectious virions at 39 degrees C, showed a normal turnover of the gag and env precursor polypeptides.     

The role of 5-S RNA in temperature-sensitive ribosomal RNA maturation.

The synthesis of 5-S RNA was found to be unchanged at both the permissive (33.5 degrees C) and non-permissive (38.5 degrees C) temperatures in a temperature-sensitive Baby Hamster Kidney cell line (BHK 21 ts 422 E) as measured relative to synthesis of 18-S rRNA. The 5-S RNA is shown to be associated with nucleolar ribonucleoprotein particles even though rRNA processing does not yield a functional 28-S rRNA at the non-permissive temperature. The amount of 5-S RNA found associated with the 80-S ribonucleoprotein particles was the same at the permissive and non-permissive temperatures, indicating that an aberrant 5-S RNA contribution to rRNA processing is not a primary cause for the temperature-sensitive lesion of rRNA maturation in this mutant cell line. The amount of 5-S RNA in nucleolar 80-S RNA particles indicated that the association of 5-S RNA with the rRNA precursor particle occurs before the cleavage step at which 32-S precursor RNA is produced.     

Characterization of a temperature-sensitive membrane alteration in chick embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus

The intramembrane particles of freeze-fractured chick embryo fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (TS68) are distributed differently at the permissive and non-permissive temperatures if, and only if, the cells are treated with glycerol before fixation. Few aggregates of intramembrane particles are present in glycerol-treated cells grown at the permissive temperature for transformation (36 degrees C), while numerous large aggregates of particles are present at the non-permissive temperature (41 degrees C). Changes in the distribution of particles after cells are shifted from 36 to 41 degrees C are observed after 20 min, while a temperature shift from 41 to 26 degrees C causes changes in glycerol-induced redistributions after 1 h. The changes observed in temperature shifts from 36 to 41 degrees C and from 41 to 36 degrees D do not require protein synthesis or RNA synthesis.     

Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles.

We have identified two temperature-sensitive peroxisome-deficient mutants of Hansenula polymorpha (ts6 and ts44) within a collection of ts mutants which are impaired for growth on methanol at 43 degrees C but grow well at 35 degrees C. In both strains peroxisomes were completely absent in cells grown at 43 degrees C; the major peroxisomal matrix enzymes alcohol oxidase, dihydroxyacetone synthase and catalase were synthesized normally but assembled into the active enzyme protein in the cytosol. As in wild-type cells, these enzymes were present in peroxisomes under permissive growth conditions (< or = 37 degrees C). However, at intermediate temperatures (38-42 degrees C) they were partly peroxisome-bound and partly resided in the cytosol. Genetic analysis revealed that both mutant phenotypes were due to monogenic recessive mutations mapped in the same gene, designated PER13. After a shift of per13-6ts cells from restrictive to permissive temperature, new peroxisomes were formed within 1 h. Initially one--or infrequently a few--small organelles developed which subsequently increased in size and multiplied by fission during prolonged permissive growth. Neither mature peroxisomal matrix nor membrane proteins, which were present in the cytosol prior to the temperature shift, were incorporated into the newly formed organelles. Instead, these proteins remained unaffected (and active) in the cytosol concomitant with further peroxisome development. Thus in H.polymorpha alternative mechanisms of peroxisome biogenesis may be possible in addition to multiplication by fission upon induction of the organelles by certain growth substrates.     

Control of protein synthesis by a temperature-sensitive mutant of reovirus 3. I. Temperature-sensitive function of ts261-b mutant.

The ability of a temperature-sensitive (ts) mutant of reovirus, ts261-b, to synthesize virus-specific RNAs and proteins during infection at the nonpermissive temperature (37 degrees C) was investigated. The relative amounts of the mutant virus-specific single-stranded (ss) RNA''s and double-stranded (ds) RNA''s synthesized in cells at 37 degrees C were 20 to 25% as much as those synthesized in the wild-type virus-infected cells. The 10 segments of the mutant ds RNAs and the three size classes of the ss RNAs were synthesized in the usual proportions. The methylation of the mutant viral mRNA''s (ss RNAs) was not blocked at 37 degrees C in infected cells. A striking temperature-sensitive restricted function of the ts261-b mutant was expressed in the synthesis of the viral proteins. This study, which uses an in vitro protein-synthesizing system reconstituted with an endogenous polysomal fraction and a postribosomal supernatant from reovirus-infected cells, has demonstrated that the endogenous polysomes obtained from ts261-b mutant-infected cells at 37 degrees C are not active in the synthesis of the viral polypeptides of known molecular weights, and the amounts of the mutant viral polypeptides synthesized in vitro by these polysomes are 5 to 9% of those synthesized by the corresponding fraction from wild-type-infected cells. The impaired protein-synthesizing capacity of the mutant virus-specific polysomes can be restored during maintenance of the infected cells at 30 degrees C after shift-down from 37 degrees C. The in vitro synthesis of viral polypeptides of known size by the active endogenous polysomes derived from cells infected at the permissive temperature is accelerated by the addition of the postribosomal supernatant obtained from cells infected at the permissive temperature. The postribosomal supernatant from mutant-infected cells at 37 degrees C did not have a stimulatory effect, but rather, it inhibited in vitro viral protein synthesis.     

Detection of an 85,000-dalton phosphoprotein in ts110 murine sarcoma virus-infected cells with antiserum against a v-mos peptide.

We have used an antiserum directed against a synthetic v-mos peptide (anti-C3 serum) to screen ts110 murine sarcoma virus (MuSV)-infected cells for the presence of v-mos-encoded proteins. Anti-C3 serum specifically recognized an 85,000-dalton protein doublet (P85) from [35S]methionine-labeled ts110 MuSV-infected producer cells grown at 32 degrees C, the permissive temperature for transformation. The P85 doublet was also recognized by an antiserum directed against the viral gag protein p15. P85 was present but at 2- to 10-fold-lower levels in ts110 MuSV-infected producer cells grown at 39 degrees C, the restrictive temperature for transformation. The P85gag-mos fusion product was the only v-mos protein reproducibly detected in this ts110 MuSV-transformed cell line. Immunoprecipitation of 32P-labeled cells with anti-C3 serum revealed that the upper band of the P85 doublet is phosphorylated, containing mostly phosphoserine and some phosphothreonine. Cells acutely infected with ts110 MuSV contained slightly higher levels of P85 than did the ts110 MuSV-infected producer cell line. Anti-C3 serum specifically recognized a 33,000-dalton protein (p33) in the acutely infected cells labeled with [35S]methionine. p33 was present in trace amounts and may represent a previously unidentified ts110 MuSV-encoded v-mos protein.     

Defect in polyamine metabolism in a BHK cell mutant temperature-sensitive for rRNA maturation

A mutant of BHK cells (ts422E) temperature-sensitive for processing 32S rRNA to 28S rRNA (Toniolo et al., '73) also loses the ability to synthesize polyamines and 5.8S rRNA when shifted to the non-permissive temperature (39 degrees). The activity of several enzymes not involved with polyamine synthesis, methylation of 32S rRNA, and small nuclear RNA production are apparently unaffected after at least 24 hours at 39 degrees. When cultures are returned to the permissive temperature (33 degrees), polyamine synthesizing capacity returns to normal as mature rRNA production resumes.     

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.     

Enhanced secretion of cell wall bound enolase into culture medium by the soo1-1 mutation of Saccharomyces cerevisiae

In order to identify the protein(s) secreted into culture medium by the soo1-1/ret1-1 mutation of Saccharomyces cerevisiae, proteins from the culture medium of cells grown at permissive (28 degrees C) and non-permissive temperatures (37 degrees C), were analyzed. Comparison of protein bands separated by SDS-PAGE identified a prominent band of 47-kDa band from a mutant grown at 37 degrees C. N-terminal amino acid sequencing of this 47-kDa protein showed high identity with enolases 1 and 2. Western blot analysis revealed that most of the cell wall-bound enolase was released into the culture medium of the mutant grown at 37 degrees C, some of which were separated as those with lower molecular weights. Our results, presented here, indicate the impairment of cell wall enolase biogenesis and assembly by the soo1-1/ret1-1 mutation of S. cerevisiae.     

Circular dichroism and ethidium bromide binding capacity of chromatin from cells temperature sensitive for the transformed phenotype.

Clone H6-15/163 is a clone of cells, originally derived from SV-40 transformed 3T3 cells, which express the transformed phenotype at low (32 degrees C) but not at high (39 degrees C) temperature. Chromatin was isolated from these cells grown at either temperature and studied by circular dichroism and for its ability to bind the intercalating dye, ethidium bromide. During the exponential phase of growth the chromatins of cells grown at either 32 or 39 degrees C are undistinguishable. Cessation of growth in confluent cultures results in marked changes in circular dichroism spectra and in ethidium bromide binding capacity of chromatin. The changes are much are much more pronounced at 39 degrees C (where the cells truly become quiescent) than at 32 degrees C (where cell proliferation continues although the number of cells per culture remains stationary). Temperature shifts and medium replacement also cause changes in chromatin structure, but the changes are again related to the extent of cell proliferation. It is concluded that the chromatin changes occurring in H6-15/163 cells and detectable by circular dichroism and ethidium bromide binding can be related to the proliferating activity of the cultured cells rather than to the expression of the transformed or untransformed phenotype.     

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.     

Poly(ADP-ribose) polymerase activity in mouse cells which exhibit temperature-sensitive DNA synthesis

The poly(ADP-ribose) polymerase activity of wild-type mouse L cells and of Balb/C-3T3 mouse fibroblasts remained relatively unchanged (at approx. 400 nmol substrate utilized/mg DNA per h) in actively-growing cells incubated at 34 degrees C or at 38.5 degrees C for at least 72 h. A similar result was obtained with the following temperature-sensitive cells grown at the permissive temperature (34 degrees C): ts A1S9 mouse L cells, ts C1 mouse L cells and Balb/C-3T3 ts mouse fibroblasts. The poly(ADP-ribose) polymerase activity of the temperature-sensitive cells was little affected during incubation for 20-24 h at the non-permissive temperature of 38.5 degrees C under which conditions temperature-inactivation of DNA replication was complete. Thereafter, this enzyme activity was found to increase some 2-fold, at a time when normal semi-conservative DNA synthesis was totally suppressed and replaced by repair replication (Sheinin, R. and Guttman, S. (1977) Biochim. Biophys. Acta 479, 105-118; Sheinin, R., Dardick, I. and Doane, F.W. (1980) Exp. Cell. Res., in the press).     

Effect of temperature on arginine incorporation by ribosomeless extracts of cells transformed by a temperature-sensitive mutant of Rous sarcoma virus.

The effect of transformation of normal rat kidney cells by a temperature-sensitive mutant of the Prague strain of Rous sarcoma virus (ts LA 24 PR-A) on the post-translational addition of arginine to the NH2-terminus of preformed acceptor molecules has been studied. Cells maintained at the permissive (35 degrees C) temperature show a high arginine-incorporating activity in ribosome free extracts compared to that found in extracts of cells grown at the non-permissive (40 degrees C) temperature. Temperature shift experiments as well as studies with cells transformed by wild type Rous sarcoma virus suggest that the decreased activity in cells grown at 40 degrees C is not due to a high temperature per se. The lower arginine incorporation in the 40 degrees C cell extracts is partially due to a decrease in the activity of arginyl transferase which catalyses the transfer of arginine from arginyl tRNA to the acceptor protein. Polyacrylamide gel electrophoresis of the radioactive product shows that the acceptor molecules present in extracts of cells grown at 40 degrees C are larger and qualitatively different from those found in extracts of cells grown at 35 degrees C.     

Characterization of temperature-sensitive mutants of simian rotavirus SA11: protein synthesis and morphogenesis.

The synthesis of viral polypeptides, distribution of viral antigens, and morphogenesis of viral structures have been examined in cells infected with temperature-sensitive (ts) mutants of SA11 representing 10 recombination groups. At the permissive temperature (31 degrees C) the synthesis of viral polypeptides and the distribution of viral antigens did not differ significantly from those of the wild type. At the nonpermissive temperature (39 degrees C) some mutants (tsB, -C, -E, -F, and -G) synthesized significantly smaller amounts of viral polypeptides and had a very diffuse distribution of viral antigen. Several of the mutants synthesized one or more electrophoretically aberrant polypeptide species at both 31 and 39 degrees C. All of the mutants, except tsF, assembled morphogenetic intermediates at 39 degrees C. Aberrant intermediates were assembled in all mutants at 31 and 39 degrees C. No specific morphogenic defect could be associated with any of the ts mutants.     

Transformation-associated changes in nuclear-coded mitochondrial proteins in 3T3 cells and SV40-transformed 3T3 cells

Comparative two-dimensional gel electrophoretic studies were performed on mitochondrial proteins in nontransformed mouse 3T3 cells and in SV40-transformed 3T3 cells, SV-T2. Two polypeptides, of 58 and 40 kDa, were present in increased amounts in SV40-transformed cells. These polypeptides were demonstrated to be nuclear-coded mitochondrial proteins by their absence in mitochondrial preparations, when labeling was performed in the presence of a mitochondrial-specific inhibitor, Rhodamine 6G. Temperature-sensitive mutants for transformation were derived from 3T3 cells by transfection with cloned SV40 DNA containing the ts A58 mutation. Increased amounts of the 58 kDa protein were apparent in these cells at the permissive temperature (33 degrees C) compared to the restrictive temperature (39.5 degrees C).