Rapid extracellular release of cytochrome c is specific for apoptosis and marks cell death in vivo.

Diverse death stimuli including anticancer drugs trigger apoptosis by inducing the translocation of cytochrome c from the outer mitochondrial compartment into the cytosol. Once released, cytochrome c cooperates with apoptotic protease-activating factor-1 and deoxyadenosine triphosphate in caspase-9 activation and initiation of the apoptotic protease cascade. The results of this study show that on death induction by chemotherapeutic drugs, staurosporine and triggering of the death receptor CD95, cytochrome c not only translocates into the cytosol, but furthermore can be abundantly detected in the extracellular medium. The cytochrome c release from the cell is a rapid and apoptosis-specific process that occurred within 1 hour after induction of apoptosis, but not during necrosis. Interestingly, elevated cytochrome c levels were observed in sera from patients with hematologic malignancies. In the course of cancer chemotherapy, the serum levels of cytochrome c in the majority of the patients grew rapidly as a result of increased cell death. These data suggest that monitoring of cytochrome c in the serum of patients with tumors might serve as a useful clinical marker for the detection of the onset of apoptosis and cell turnover in vivo.

Ribonucleotide reductase R2 gene expression and changes in drug sensitivity and genome stability.

Ribonucleotide reductase is a highly regulated, cell cycle-controlled activity that plays an important role in DNA synthesis and repair. Recent studies have shown that elevated expression of the rate-limiting R2 component of ribonucleotide reductase increases Raf-1 protein activation and mitogen-activated protein kinase activity and acts as a novel malignancy determinant in cooperation with activated oncogenes like H-ras. We show that hydroxyurea-resistant mouse L cells with elevated R2 gene expression and increased ribonucleotide reductase activity exhibit significantly decreased sensitivities to the chemotherapeutic compounds N-(phosphonacetyl)-L-aspartate (PALA) and methotrexate (MTX). Furthermore, BALB/c 3T3 cells containing a retroviral expression vector encoding the R2 sequence also showed decreased sensitivity to PALA and MTX when compared to cells containing the same vector but without the R2 coding region. Colonies that developed in the presence of PALA or MTX contained amplifications of the CAD or dihydrofolate reductase genes and exhibited wild-type p53 function as determined in sequence-specific p53 binding activity assays. NIH-3T3 cells containing the R2 retroviral expression vector also showed significantly decreased sensitivity to hydroxyurea and MTX but not to PALA. Furthermore, NIH-3T3 cells transfected with a vector containing the R2 sequence in antisense orientation exhibited increased sensitivity to hydroxyurea, PALA, and MTX. Similarly, mouse 10T1/2 cells that are highly transformed and drug resistant due to alterations in H-ras and a mutant oncogenic form of p53 exhibited significant increases in sensitivity to hydroxyurea, PALA, and MTX when transfected with a vector containing the R2 sequence in antisense orientation and compared to cells containing the same vector without the antisense sequence. These results indicate that altered expression of the R2 component is capable of significantly modifying drug sensitivity properties of tumor cells. We hypothesize that this occurs, at least in part, through a mechanism of increased genetic instability that is independent of direct p53 mutation or loss and involves R2 stimulation of the mitogen-activated protein kinase signal pathway.

2',3'-Cyclic nucleotide 3'-phosphodiesterase binds to actin-based cytoskeletal elements in an isoprenylation-independent manner.

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is an isoprenylated protein enriched in myelin and oligodendrocytes but also present in several other tissues at low levels. CNP binds avidly to membranes and in addition possesses several characteristics of cytoskeletal proteins. The role of isoprenylation in the association of CNP with the cytoskeleton was analyzed by ectopic expression in L cells of epitope-tagged CNP1 and a non-isoprenylated mutant CNP1. Using nonionic detergent extraction, drug-mediated cytoskeletal disruption, and coimmunoprecipitation with an anti-actin antibody, we show that CNP1 is associated with actin-based cytoskeletal elements independently of its isoprenylation status. A control protein, p21c-H-ras, which is also modified by isoprenylation at its carboxyl-terminus, does not bind to cytoskeletal structures as judged by the same criteria. We present a model that accounts for the association of CNP1 with membranes and the cytoskeleton.

Subcellular localization and targeting of cathepsin E.

The subcellular distribution and targeting of the non-lysosomal aspartic proteinase cathepsin E have been studied using mouse L cells and monkey Cos 1 cells that were transfected with cDNA encoding cathepsin E. The cathepsin E was retained in L cells for at least 20 h without significant degradation and its single N-linked oligosaccharide remained sensitive to endo-beta-N-acetyl-glucosaminidase H. When cathepsin E was overexpressed by transient transfection in Cos 1 cells, it was very slowly secreted into the media. The intracellular form of the enzyme contained a high mannose oligosaccharide which was processed to a complex type species upon secretion. In double label immunofluorescence studies, cathepsin E co-localized with cathepsin D-myc-KDEL, an endoplasmic reticulum (ER) marker. Subcellular fractionation on a Percoll density gradient showed that the cathepsin E co-migrated with membranous vesicles that were distinct from dense lysosomes. Only a trace amount of the enzyme was recovered in the soluble fraction. These findings indicate that in L cells and Cos 1 cells, the intracellular location of cathepsin E is the endoplasmic reticulum. To identify the protein sequences required for ER retention, we made chimeric proteins between cathepsin E and pepsinogen, an aspartic proteinase that is rapidly secreted by Cos 1 cells. We found that amino acids 1-48 of cathepsin E are important for its retention in the ER. Within this region, Cys7, which is involved in covalent dimer formation, plays a significant role in the retention.

Effect of cell line on in vitro metal ion cytotoxicity.

OBJECTIVES: The choice of cell line for in vitro biological tests which assess the cytotoxicity of dental materials remains controversial, yet this issue is important because these tests are widely used to rate the biocompatibility of new and existing materials, and many different cell lines are commonly used. The purpose of the current study was to quantify the responses of four cell lines (Balb/c 3T3, L929, ROS 17/2.8 and WI-38) to 14 metal ions which are released from dental materials, and relate these responses to the metabolic activity and population doubling times of these cells. METHODS: Succinic dehydrogenase (SDH) activity was used to monitor metabolic activity and cytotoxic response. RESULTS: The cell lines responded differently to most metal ions. In general, the Balb/c 3T3 line was the most sensitive, and the WI-38 line was the least sensitive. However, there were many exceptions depending on the metal ion. The passage number of the cells also affected the cytotoxic response. It was concluded that the cytotoxicity of materials which release metal ions will be significantly different depending on which cell line is selected and its passage number. SIGNIFICANCE: Based on the findings that cell lines ranked the toxicities of the metal ions similarly, it seems reasonable to use these types of in vitro tests to rank the cytotoxicities of materials. However, if these types of tests are used to predict in vivo cytotoxicity, care should be taken to choose conditions and cells which are relevant.

Cells enriched for catalase are sensitized to the toxicities of bleomycin, adriamycin, and paraquat.

L cells were enriched about 100-fold for catalase by the transcription of the transfected cDNA for human catalase. In spite of substantial enrichment for catalase, the transfected cells (LFN7C/B3) were not resistant to the cytotoxicity of a variety of agents which reduce dioxygen, including paraquat, menadione, adriamycin, phenazine methosulfate, and bleomycin. Instead, they were more sensitive to paraquat, bleomycin, and adriamycin than the untransfected cells from which they derived. Desferrioxamine afforded the cells enriched for catalase modest protection from the toxicities of bleomycin, paraquat, or adriamycin, suggesting that enrichment for iron as a consequence of the increased cellular content of catalase accounted for some of the increased sensitivities. The increased sensitivity of the LFN7C/B3 cells to bleomycin and paraquat was also attributed to the ability of catalase in cells to prevent the drug-induced consumption of O2; by capturing H2O2 before it can escape the cell and converting it to O2, catalase can maintain the concentration of O2 either for repeated rounds of chemical reduction or for direct interaction with the toxin. As predicted by this formulation, the toxicities of paraquat and of bleomycin were increased in an atmosphere of 40% dioxygen in comparison with an atmosphere of 20% dioxygen.

Differing effects of the inhibition of poly(ADP-ribose) polymerase on the course of oxidative cell injury in hepatocytes and fibroblasts.

The effects of the two inhibitors of poly(ADP-ribose) polymerase, 3-aminobenzamide (ABA) and benzamide (BA), on the oxidative killing of L929 mouse fibroblasts and primary cultures of rat hepatocytes were studied. The killing of L929 cells by tert-butyl hydroperoxide (TBHP) occurred by two mechanisms, one sensitive and the other insensitive to the antioxidant N,N'-diphenylphenylene diamine (DPPD). Cell killing by either mechanism was prevented by the ferric iron chelator deferoxamine. ABA and BA prevented the killing of L929 cells that occurred in the presence, but not in the absence, of DPPD. ABA and BA inhibited the activity of poly(ADP-ribose) polymerase by 85%. Protection was accompanied by the sparing of the depletion of both NAD and ATP, but there was no effect of either ABA or BA on the iron-dependent appearance of single-strand breaks in DNA. Depletion of ATP by treating the fibroblasts with 2-deoxyglucose and sodium azide did not result in any loss of viability. H2O2 similarly killed the L929 cells by a mechanism that depended on a source of ferric iron. However, DPPD had no effect on the cell killing, and ABA and BA completely protected the cells in the presence or absence of DPPD. H2O2 caused the appearance of single-strand breaks that were prevented by deferoxamine, but again not by ABA or BA. ABA and deferoxamine reduced, but did not prevent, the depletion of both NAD and ATP occurring with H2O2. With the cultured hepatocytes, ABA and BA inhibited poly(ADP-ribose) polymerase at concentrations that were without effect on either the extent of cell killing or the depletion of NAD occurring with either TBHP, H2O2, or menadione. These data indicate that the relationship between oxidative DNA damage and the genesis of lethal injury is very different in the two types of cells. In the fibroblasts, the appearance of single strand breaks in DNA was accompanied by depletion of NAD and ATP and subsequently by the death of the cells. These events were mediated by the activity of poly(ADP-ribose) polymerase, as inhibition of the enzyme prevented their development. In the hepatocytes, inhibition of poly(ADP-ribose) polymerase was without effect on the oxidative death of the cells.

Correlation among oxysterol potencies in the regulation of the degradation of 3-hydroxy-3-methylglutaryl CoA reductase, the repression of 3-hydroxy-3-methylglutaryl CoA synthase and affinities for the oxysterol receptor.

25-Hydroxycholesterol regulates cholesterol biosynthesis by two mechanisms: repression of the transcription of the genes for several cholesterogenic enzymes and acceleration of the degradation of the enzyme 3-hydroxy-3-methylglutaryl CoA reductase. In the present work the structural features which govern oxysterol potency were determined separately for each regulatory mechanism. Regulation of degradation was tested using a 3-hydroxy-3-methylglutaryl CoA reductase-beta-galactosidase fusion protein. Repression of enzyme synthesis was tested by measuring 3-hydroxy-3-methylglutaryl CoA synthase activity since this protein is not regulated by a degradative mechanism. Oxysterol activities were highly correlated between the two assays (R = .959) demonstrating that the degradative and repressor mechanisms share an element which determines oxysterol regulatory potency. Correlation of these results with previous data for the affinity of these oxysterols for the oxysterol receptor suggests that the receptor is the element involved in both these regulatory pathways.

Characterization of a second promoter for the mouse liver/bone/kidney-type alkaline phosphatase gene: cell and tissue specific expression.

The second leader exon and the relative promoter of the mouse liver/bone/kidney-type alkaline phosphatase gene were identified and characterized. The transcription initiation site was determined by S1 mapping analysis. The differential expression of the two alternatively spliced transcripts was assessed in cell lines of different origin and in various tissues by polymerase chain reaction and RNase mapping analysis. The first promoter is active in embryo derived cells, whereas the second promoter is silent in basal conditions but it is activated by dibutyryl cAMP in fibroblastic cells. In the whole animal, the transcript driven by the first promoter is found in most tissues albeit at different levels, while the one driven by the second promoter is specifically expressed at high levels only in the heart.

Effect of coherence time of the applied magnetic field on ornithine decarboxylase activity.

Skepticism over the possibility of weak electromagnetic fields affecting cell function exists because endogenous thermal noise fields are larger than those reported to cause effects. Four-hour exposure to a 55- or 65-Hz field approximately doubles the specific activity of ornithine decarboxylase (ODC) in L929 cells. To test the idea that the cell discriminates against this thermal noise because it is incoherent, partial incoherence was introduced into the applied field by shifting the frequency between 55- to 65-Hz at intervals of tau coh--delta tau where tau coh is a predetermined time interval and delta tau much less than tau coh varies randomly from one frequency shift to the next. To obtain the full ODC enhancement, coherence of the impressed signal must be maintained for a minimum of about 10s. For tau coh = 5.0s a partial enhancement is elicited, and at 1.0s there is no response. Unfortunately coherence times of this duration are too short to solve the thermal noise puzzle.

Cell cycle regulation of thymidine kinase: residues near the carboxyl terminus are essential for the specific degradation of the enzyme at mitosis.

The level of human thymidine kinase (TK) polypeptide is subject to cell cycle regulation. The enzyme is barely detectable in G1 phase but increases 10- to 20-fold by M phase. The low level of human TK in G1 phase is due primarily to the specific degradation of the protein during cell division. Substitution of heterologous promoters, removal of the introns, and deletion of all of the 3' untranslated region from the human TK gene do not affect cell cycle regulation of the enzyme. However, deletion of the carboxyl-terminal 40 amino acids or fusion of beta-galactosidase to the carboxyl terminus of human TK completely abolishes cell cycle regulation and stabilizes the protein throughout the cell cycle. These alterations do not significantly alter the specific enzymatic activity of TK. Changing the carboxyl terminus or deletion of the last 10 amino acids does not alter cell cycle regulation. These data demonstrate that residues near the carboxyl terminus of TK are essential for the cell cycle phase-specific degradation of the enzyme.

Ouabain- and Ca2(+)-sensitive ATPase activity of chimeric Na- and Ca-pump molecules.

Chimeric ion-pumps, consisting of the N-terminal 2/3 of the alpha 1-subunit of the ouabain-sensitive chicken Na+,K(+)-ATPase and the C-terminal 1/3 of the sarcoplasmic reticulum Ca2(+)-ATPase, were expressed in ouabain-insensitive mouse L cells. These chimeric molecules exhibited ouabain-sensitive ATPase activity very similar to that of the wild-type chicken Na+, K(+)-ATPase. This ATPase activity could be stimulated by adding Ca2+ to the assay system. These results suggest that the sites for ouabain-inhibition are restricted to the N-terminal 2/3 of the Na-pump, and the C-terminal 1/3 of the Ca-pump interacts with Ca2+.

After X-irradiation a transient arrest of L929 cells in G2-phase coincides with a rapid elevation of the level of O6-alkylguanine-DNA alkyltransferase.

Following X-irradiation of exponentially growing L929 cells two major phenomena have been observed. First, there was a delay in cell division which can be ascribed to the arrest of cells in the G2-phase (G2-block), and, second, the cellular content of the O6-alkylguanine-DNA alkyltransferase (AGT) was markedly increased. Flow cytometrical DNA-measurements revealed that cells began to accumulate in the G2-phase 4 h after irradiation (p.r.) irrespective of the X-ray dose, while both the fraction of cells blocked in G2 and the time period the cells persisted in G2 increased with the radiation dose. About 24 h past release from the G2-block the distribution of cells in the cell cycle was similar to that of untreated control cells. In comparison with control cells the AGT content in irradiated cells (4 Gy) was highest at about 48 h p.r. (3.4-fold increase). The highest ratio of increase in AGT was, however, observed to occur between about 4 and 13 h p.r. (2.6-fold increase). As shown by flow cytometrical measurements using a BrdUrd/DNA double labeling technique, this rapid primary increase in AGT coincides very well with the entrance of cells into the G2-phase. This indicates that the cellular AGT content in X-irradiated (parental) cells started to exceed the basal level at the beginning of the G2-phase, but not before or during the S-phase. Once the AGT level was elevated it continued to increase for 2 to 3 cell doubling times.

Inhibition of transient gene expression with plasmids encoding herpes simplex virus type 1 UL55 and alpha genes.

Herpes simplex virus type 1 (HSV-1) subgenomic sequences from 0.743 to 0.782 map units have been molecularly cloned as plasmid AT1 and shown to inhibit stable DNA-mediated gene transformation of Ltk- cells with the HSV-1 thymidine kinase (tk) gene. Here it is shown that AT1 also inhibits transient gene expression. Expression from the chloramphenicol acetyltransferase (CAT) gene under the control of either the HSV-1 tk gene or the Rous sarcoma virus (RSV) promoter was inhibited when cotransfected into Ltk- and CV-1 cells with equimolar amounts of AT1. AT1 was subcloned as three overlapping plasmids called AT1a, alpha 27 and AT1b. The alpha 27 plasmid encodes the HSV-1 immediate early gene, alpha 27; AT1a possesses sequences that specify an open reading frame in HSV-1 strain KOS used in these studies, although the significance of this open reading frame is unknown; AT1b possesses the sequences for UL55 and UL56, also genes for which no function has been reported. No single subclone or pair of subclones demonstrated significant inhibition of transient gene expression. Cotransfection of all three subclones did result in inhibition of RSV-CAT gene expression, suggesting that information from each subclone is necessary. One of the three subclones, alpha 27, contains the HSV-1 immediate early gene, alpha 27, so the possibility that other immediate early genes could substitute for alpha 27 was tested. Inhibition of RSV-CAT gene expression was also achieved by cotransfection of AT1a and AT1b with either an alpha 0- or alpha 4-containing plasmid, suggesting that the role of the alpha 27-containing plasmid can be replaced by other alpha genes with trans-regulating capability. Finally, AT1a and AT1b linker insertion mutants have been constructed and used to study the role these plasmids play in mediating inhibition. These results suggest that AT1 contains HSV-1 functions in addition to that of alpha 27 that interfere with gene expression.

Effect of protein kinase C inhibitors on interferon-beta production by viral and non-viral inducers.

Induction of interferon-beta (IFN-beta) in human (BG-9), simian (CV-1) and mouse (L-929) cell lines by Sendai virus and by poly(rI). poly(rC) has been studied for its possible dependence on protein kinase C (PKC) through the use of pharmacological inhibitors (K252a and H-7) of PKC. Exposure of BG-9, CV-1 or L-929 cells to K252a (greater than or equal to 0.025 microM), a staurosporine derivative, 24 h before or after induction of IFN with poly(rI).poly(rC), inhibited by greater than 95% the production of IFN-beta. In contrast, virus-induced IFN production was enhanced threefold or more by K252a in BG-9 and L-929 but not in CV-1 cells. A naphthalene sulphonamide inhibitor of PKC, H-7, at greater than or equal to 5 microM, decreased poly(rI).poly(rC)-induced IFN production in BG-9 and CV-1 cells by 75 to 94%, but had no effect on IFN production in L-929 cells. Viral induction of IFN was not affected significantly by H-7 in BG-9, CV-1 and L-929 cells. In contrast to these results, the calmodulin inhibitor, trifluoperazine (5 to 15 microM) did not affect IFN-beta production by poly(rI).poly(rC) but significantly enhanced IFN production by Sendai virus in both human and murine cell lines. Thus, in human and simian fibroblasts the induction of IFN-beta by poly(rI).poly(rC) appears to be PKC-dependent, whereas viral induction of IFN-beta is not. Results with K252a implicate PKC in non-viral induction of IFN in mouse fibroblasts, as well. Direct measurements of PKC activity in BG-9 cells exposed to several concentrations of K252a showed that the membrane PKC activity is significantly more sensitive to inhibition by K252a than is cytosolic PKC activity. In L-929 cells, K252a inhibited membrane PKC activity similarly, but was less effective as an inhibitor of cytosolic enzyme activity than in BG-9. These studies support an integral role for PKC activity, particularly membrane-associated activity, in non-viral [poly(rI).poly(rC)] induction of IFN-beta in human, simian and mouse fibroblasts.

Expression pattern of the hepatitis B virus genome in transfected mouse fibroblasts.

Permanent mouse fibroblast LTK- cells were transfected with dimeric hepatitis B virus (HBV) DNA linked to the simian virus 40 (SV40) early promoter/enhancer. Many clones stably expressed high levels of polyadenylated RNAs encoding hepatitis B surface (HBs) proteins (2.1 kb), HBe protein (3.6 kb), and HBx protein (0.6 kb). Although a chimeric RNA (4.0 kb) probably starting from the SV40 promoter was also synthesized, transcription of viral RNAs was predominantly directed by HBV promoters and its terminator. In contrast to HBV-transfected liver cells, the fibroblasts expressed only pregenomic 3.6-kb transcripts starting 5' to, but not within, the precore sequence. Thus, no normal core protein could be synthesized, but the cells expressed and secreted HBe protein of heterogeneous size. Small and middle HBs proteins were strongly expressed, while large HBs protein was almost absent. HBx mRNA expression was more efficient in mouse fibroblasts than in human hepatoma cells and 18-kDa HBx protein was exclusively detected in purified nuclei. Expression of HBe, small and middle HBs, and HBx proteins apparently does not require hepatic factors. Underexpression of HBc mRNA and large HBs mRNA suggests that activity of their promoters depends on cell-type-specific transcription factors.

Replication of hepatitis B virus in transfected nonhepatic cells.

Permanent murine fibroblasts (LTK-) were transfected with a dimer of hepatitis B virus (HBV) DNA and a neomycin resistance gene which were both linked to the simian virus 40 (SV40) early promoter/enhancer. One of the stably transfected clones, LTK4/36, which secreted HBsAg, HBeAg, and HBV DNA was further analyzed. It contained eight to nine copies of integrated HBV DNA per haploid genome and low amounts of episomal HBV DNA. The secreted viral DNA was covalently linked to protein and was associated with particles which had the characteristic density of natural virions from serum of human viremic carriers. The particles contained an endogenous DNA polymerase, small and middle surface proteins, but in contrast to natural virions very little core protein and large surface protein. Instead of core protein, they contained incompletely processed HBe protein which is colinear to core protein. The fibroblast-derived virions were less stable than virions from human carriers or from transfected hepatoma cells. After several days of storage, their DNA was only partially protected against DNase. Obviously, nonhepatic cells can express HBV-like particles, even if liver-dependent gene products like large surface protein and core protein are missing.

Illegitimate and homologous recombination in mammalian cells: differential sensitivity to an inhibitor of poly(ADP-ribosylation).

We determined the effect of 3-methoxybenzamide (3-MB), a competitive inhibitor of poly(ADP-ribose)polymerase (E.C. 2.4.2.30), on illegitimate and extrachromosomal homologous recombination in mouse Ltk- cells. Cells were transfected with a wild type Herpes thymidine kinase (tk) gene or with two defective tk gene sequences followed by selection for tk-positive colonies. Using a wild type tk gene, colony formation required uptake, integration, and expression of the tk gene. Using defective tk genes, colony formation had the additional requirement for homologous recombination to reconstruct a functional tk gene. The presence of non-cytotoxic levels of 3-MB during and after transfection reduced the number of colonies recovered with a wild type tk gene in a dose-dependent manner, with 2 mM 3-MB causing a 10 to 20-fold reduction. 3-MB reduced the number of colonies recovered with defective tk genes only to the same extent as in transfections with a wild type gene. Treatment with 3-methoxybenzoic acid, a non-inhibitory analog of 3-MB, did not reduce the recovery of colonies in any experiment. Similar results were obtained using linear or supercoiled molecules and when defective tk genes were transfected into cells on one or two different DNA molecules. By assaying for transient expression of the tk gene, we found that 3-MB did not inhibit uptake or expression of the tk gene. We conclude that poly(ADP-ribosylation) plays a role in random integration (illegitimate recombination) of DNA but does not play an important role in extrachromosomal homologous recombination, demonstrating that these two recombination pathways in cultured mouse fibroblasts are biochemically distinct.

DNA polymerase-primase complex in wild-type and ts A1S9 mouse L-cells, temperature-sensitive for DNA replication during cell cycle progression.

ts A1S9 mutant cells, derived from wild type WT-4 mouse L-cells, are temperature-sensitive (ts) for DNA synthesis and cell division. We try to determine the cause of the arrest of DNA replication in ts A1S9 cells at the nonpermissive temperature by comparing the modifications induced by the shift of temperature on the activity and the synthesis of DNA polymerase-alpha and DNA primase as a function of time. Forty-seven hours after temperature upshift DNA polymerase-alpha activity of ts A1S9 cells was inhibited by 90% while primase activity was barely detectable. By contrast, the activities of both enzymes increased to a plateau level in WT-4 cultured at either temperature and in ts A1S9 cells grown at the low permissive temperature. Study of the synthesis of DNA polymerase-alpha primase and of the structure of the enzyme complex during cell cycle progression was approached by immunoprecipitation of [35S]-labelled cells, with a specific monoclonal antibody directed against DNA polymerase-alpha. We have found that, irrespective of temperature of cultivation of WT-4 or ts A1S9 cells, this antibody precipitated polypeptides of 220, 186, 150, 110, 68-70, 60, and 48 kDa from cell extracts. With ts A1S9 cells cultivated at 38.5 degrees C for 48 hr the polypeptides of 220 and 186 kDa, associated with alpha-polymerase activity, were considerably more abundant than in the control cells, with a concomitant decline in the polypeptides of 60 and 48 kDa, implicated in primase activity. Thus the inhibition of DNA polymerase-alpha cannot be due to a decreased synthesis of the 186 kDa subunit but to its temperature inactivation. Consistent with a recent asymmetric dimeric model where polymerase-alpha complex and polymerase delta complex synthesize co-ordinately at the replication fork lagging and leading DNA strands, the observed alterations of polymerase-alpha and primase content explain the inhibition of DNA synthesis and the cell cycle arrest of the ts A1S9 cells at the nonpermissive temperature.

Transfection of DHFR- and DHFR+ mammalian cells using methotrexate-resistant mutants of mouse dihydrofolate reductase.

Site-directed mutagenesis was used to generate mutants of mouse dihydrofolate reductase more resistant to methotrexate than the wild type enzyme. The mutant genes were used to transfect either DHFR- or DHFR+ cell lines. These mutants, as well as the wild type gene, were able to confer methotrexate resistance to DHFR- CHO cells. The number of selected colonies decreased with increased concentrations of methotrexate. The number of colonies observed at 10 microM methotrexate is correlated with the Ki(MTX) of the enzyme: the higher the Ki, the higher the number of colonies for the corresponding mutant. In contrast, the transfection of DHFR+ cells gave a few numbers of colonies not different for the wild type and the mutants.