Inhibition of dihydrofolate reductase gene expression following serum withdrawal or db-cAMP addition in methotrexate-resistant mouse fibroblasts

We have used a methotrexate (MTX)-resistant mouse 3T6 cell line (M50L3), that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of 300, to study the mechanism for turning off DHFR gene expression following withdrawal of serum factors or elevation of the intracellular level of cAMP. When resting (G0) M50L3 cells are serum-stimulated to reenter the cell cycle, the level of DHFR activity begins to increase at about the same time the cells begin synthesizing DNA. The increase in enzyme activity is preceded by increases in the synthesis rate of the enzyme, and the content and production rate of DHFR mRNA. These increases, as well as entry into S phase, are blocked when the cells are serum-stimulated in the presence of dibutyryl cyclic AMP (db-cAMP) and theophylline. In this study, we found that when these drugs were added, or the serum stimulus was withdrawn during S phase (20 h following stimulation), the subsequent increase in DHFR level was blocked. Immunoprecipitation of DHFR from pulse-labelled cells showed that both treatments led to a rapid decrease in synthesis rate of the enzyme. The effect on total protein synthesis was much less than on DHFR synthesis. In DNA-excess filter hybridization experiments, we found that the content of cytoplasmic DHFR mRNA decreased in parallel with the synthesis rate of the enzyme. This was due in part to a decrease in the production rate of DHFR mRNA relative to total mRNA. In addition, drug addition or serum withdrawal led to a significant destabilization of DHFR (as well as total) mRNA. About 85% of poly(A)(+) DHFR mRNA was associated with polysomes in resting, growing, or cAMP-treated cells, suggesting that DHFR gene expression was not controlled at the translational level under these conditions.     

High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR.

By the use of a highly sensitive mapping procedure allowing the identification of the start sites of DNA replication in single-copy genomic regions of untreated, exponentially growing cultured cells (M. Giacca, L. Zentilin, P. Norio, S. Diviacco, D. Dimitrova, G. Contreas, G. Biamonti, G. Perini, F. Weighardt, S. Riva, and A. Falaschi, Proc. Natl. Acad. Sci. USA 91:7119-7123, 1994), the pattern of DNA replication of the Chinese hamster dihydrofolate reductase (DHFR) gene domain was investigated. The method entails the purification of short stretches of nascent DNA issuing from DNA replication origin regions and quantification, within this sample, of the abundance of different adjacent segments by competitive PCR. Distribution of marker abundance peaks around the site from which newly synthesized DNA had emanated. The results obtained by analysis of the genomic region downstream of the DHFR single-copy gene in asynchronous cultures of hamster CHO K1 cells are consistent with the presence of a single start site for DNA replication, located approximately 17 kb downstream of the gene. This site is coincident with the one detected by other studies using different techniques in CHO cell lines containing an amplified DHFR gene domain.     

Cell cycle-dependent DHFR and t-PA production in cotransfected, MTX-amplified CHO cells revealed by dual-laser flow cytometry

The cell cycle-dependent regulation of the cellular dihydrofolate reductase content (DHFR) and tissue plasminogen activator (t-PA) production and secretion in plasmid-amplified cells was investigated in the DHFR-negative CHO cells transfected with the plasmid pSV-tPA.dhfr. This plasmid, carrying the dhfr and t-PA gene under control of different promotors, was amplified by serial passages in 5 microM methotrexate (MTX) for dhfr gene amplification. The intracellular amount of DHFR was quantitated in viable cells by MTX-FITC labeling and flow cytometric analysis of the FITC fluorescence. In comparison with the original CHO cells, the pSVtPA.dhfr-amplified cells showed a greater than 230-fold increase in MTX-FITC fluorescence. Using dual laser flow cytometry (uv: vital cell cycle with Hoechst 33342; 488 nm: DHFR with MTX-FITC), we show a maximum increase in the intracellular DHFR content during G1 and/or at G1/S transition (100 to 157%), followed by a continuous increase to 200% during S and G2/M. To determine t-PA production CHO cells were sorted from G1-, early/late S-, and G2/M-phase. After 1-, 2-, and 4-h incubation periods, t-PA production was quantitated using a sensitive t-PA ELISA technique. We found that t-PA production and secretion (2-h assay), unlike the expression of DHFR, increased continuously from relatively 100% in G1 to 127% in early S and reached its maximum of 159% in late S, whereas in G2/M-phase it decreased to 118%. Our results show that in pSVtPA.dhfr-coamplified CHO cells gene products DHFR and t-PA both exhibit different cell cycle-correlated accumulation and secretion, respectively, indicating that the brightest MTX-FITC-positive cells (G2/M) do not display the highest t-PA secretion rate.     

Cytophotometric measurement of the cellular DNA content of [3H]thymidine-labelled spheroids. Demonstration that some non-labelled cells have S and G2 DNA content

Spheroids from the V279-171b and MCa-11 cell lines were incubated continuously for 24 hr in [3H]thymidine for labelling of the outer cells of the viable rim. The spheroids were dispersed into single cells, and the DNA content of photomapped cells was measured by absorption cytophotometry. Autoradiographs were then prepared from which we ascertained cellular labelling. For spheroids of both cell lines, we found a larger proportion of cells with a G0/G1 DNA content among the non-labelled inner spheroid cells than among the labelled outer cells (P less than 0.001). This block of non-labelled spheroid cells in G0/G1 was not a cell cycle perturbation caused by the isotope for the MCa-11 spheroids. Approximately 8% of non labelled MCa-11 spheroid cells had S/G2 DNA content, suggesting that non-cycling cells in spheroids may be blocked in S and G2 as well as in the G0/G1 phase of the cell cycle.     

Cell-cycle studies by multiparametric automatic scanning of topographically preserved cells in culture

The authors have developed a new methodology for characterizing in situ the cell cycle of human mammary epithelial cell lines. Using a SAMBA 200 cell image processor (scanning cytometry), 15 densitometric and textural parameters were computed on each Feulgen-stained nucleus. Parameters computed from the grey level cooccurrence and run-length section matrices allowed assessment of the chromatin pattern. Multiparametric analysis of data defined: 1) the relative position of each cell; 2) the relative positions of groups of cells, each group corresponding to a definite phase of the cell cycle; and 3) the function of these parameters best separating these phases. Files then were constructed for each phase: G0/G1, S, G2/ and M. Using these three files as a reference to classify cells, it was possible to ascertain the phase of the cell cycle for each cell of a population. The MDA AG human cell line synchronized by mitotic selection was used as a model to develop this method. The criteria used to assign cells to G0/G1, S, or G2 was DNA content. Classification in M phase was achieved by visual identification of mitotic cells. This method was checked on unsynchronized MDA AG and then applied to other human cell lines (MDA MB231, MCF-7, T47D C111). Comparison of results obtained by scanning cytometry and flow cytometry showed the proportion of cells assigned to G0/G1, S, and G2/M by the two methods to be similar. This new method removes some of the limitations of flow cytometry by 1) allowing visual verification of each cell analyzed; 2) lowering the number of cells required for study; 3) discriminating between G2 and M; and 4) preserving cell topography.     

Radiation effects on DNA synthesis in a defined chromosomal replicon.

It has recently been shown that the tumor suppressor p53 mediates a signal transduction pathway that responds to DNA damage by arresting cells in the late G1 period of the cell cycle. However, the operation of this pathway alone cannot explain the 50% reduction in the rate of DNA synthesis that occurs within 30 min of irradiation of an asynchronous cell population. We are using the amplified dihydrofolate reductase (DHFR) domain in the methotrexate-resistant CHO cell line, CHOC 400, as a model replicon in which to study this acute radiation effect. We first show that the CHOC 400 cell line retains the classical acute-phase response but does not display the late G1 arrest that characterizes the p53-mediated checkpoint. Using a two-dimensional gel replicon-mapping method, we then show that when asynchronous cultures are irradiated with 900 cGy, initiation in the DHFR locus is completely inhibited within 30 min and does not resume for 3 to 4 h. Since initiation in this locus occurs throughout the first 2 h of the S period, this result implies the existence of a p53-independent S-phase damage-sensing pathway that functions at the level of individual origins. Results obtained with the replication inhibitor mimosine define a position near the G1/S boundary beyond which cells are unable to prevent initiation at early-firing origins in response to irradiation. This is the first direct demonstration at a defined chromosomal origin that radiation quantitatively down-regulates initiation.     

Macrophage-mediated cytostatic activity blocks lymphoblast cell cycle progression independently in both G1 phase and S phase

Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.     

Impact of DNA demethylation of the G0S2 gene on the transcription of G0S2 in squamous lung cancer cell lines with or without nuclear receptor agonists

We recently identified that DNA methylation of the G0S2 gene was significantly more frequent in squamous lung cancer than in non-squamous lung cancer. However, the significance of G0S2 methylation levels on cancer cells is not yet known. We investigated the effect of G0S2 methylation levels on cell growth, mRNA expression, and chromatin structure using squamous lung cancer cell lines and normal human bronchial epithelial cells. DNA methylation and mRNA expression of G0S2 were inversely correlated, and in one of the squamous lung cancer cell lines, LC-1 sq, G0S2 was completely methylated and suppressed. Overexpression of G0S2 in LC-1 sq did not show growth arrest or apoptosis. The G0S2 gene has been reported to be a target gene of all-trans retinoic acid and peroxisome proliferator-activated receptor agonists. We treated LC-1 sq with 5-Aza-2′-deoxycytidine, Trichostatin A, all-trans retinoic acid, Wy 14643, or Pioglitazone either alone or in combination. Only 5-Aza-2′-deoxycytidine restored mRNA expression of G0S2. Chromatin immunoprecipitation revealed that histone H3 lysine 9 was methylated regardless of DNA methylation or mRNA expression. In summary, mRNA expression of G0S2 was regulated mainly by DNA methylation in squamous lung cancer cell lines. When the G0S2 gene was methylated, nuclear receptor agonists could not restore mRNA expression of G0S2 and did not show any additive effect on mRNA expression of G0S2 even after the treatment with 5-Aza-2′-deoxycytidine.     

Gene amplification accompanies low level increases in the activity of dihydrofolate reductase in antifolate-resistant Chinese hamster lung cells containing abnormally banding chromosomes

Three independently-derived, antifolate-resistant Chinese hamster lung cell lines that exhibit low level increases in dihydrofolate reductase (DHFR) activity, i.e., three- to fivefold vs. controls, have been compared with drug-sensitive cells to determine relative DHFR gene content. With a solution hybridization technique that makes use of genomic DNA and a cloned double-stranded Chinese hamster DHFR cDNA probe, it has been found that the enzyme activity increases are associated with an approximately proportionate amplification of DHFR genes. Trypsin-Giemsa staining techniques and hybridizations in situ further show that the amplified DHFR genes are located within abnormally banding regions along chromosome 2q and also suggest that, in each subline, only one chromosome 2 homolog is initially involved in the amplification process.     

The pattern of dihydrofolate reductase expression through the cell cycle in rodent and human cultured cells

We have examined the pattern of dihydrofolate reductase (DHFR) enzyme and mRNA levels in cell cycle stage-specific populations obtained by centrifugal elutriation in Chinese hamster ovary cells and in a derivative line in which the dihydrofolate reductase gene is amplified approximately 50-fold. On a per cell basis, we observed a 2-fold increase in DHFR activity as cells progressed from G1 to G2/M with a concomitant 2-fold increase in the rate of protein synthesis and steady state level of mRNA. Analysis of DHFR mRNA levels in cell cycle stage-specific mouse 3T6 and human 143 tk- cells gave a similar pattern. We also demonstrate that simple alterations in growth conditions prior to elutriations can dramatically increase the levels of DHFR mRNA in all cell cycle states, thereby indicating that growth response associated with the DHFR gene functions independent of the cell cycle. We conclude that during periods of exponential growth the increases in dihydrofolate reductase activity, rate of protein synthesis, and steady state levels of mRNA parallel the general increases in cell volume and protein content associated with normal progression through the cell cycle, and therefore DHFR cannot be considered a cell cycle-regulated enzyme.     

Coregulation of dihydrofolate reductase and thymidylate synthase in overproducer cell lines of wild carrot

Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities are associated with a 285,000 molecular weight enzyme complex in carrot (Daucus carota L.). Selection for methotrexate (MTX) resistance by stepwise increase of the concentration of MTX results in a high frequency adaptation to MTX with little or no significant increase in DHFR activity. However, when as a second step following MTX selection a specific inhibitor of TS, 5-fluoro-2-deoxyuridine was used, DHFR overproducer lines were obtained. The overproduction phenotype of the lines was almost completely lost after 8 weeks of growth in the absence of selection pressure. Although DHFR and TS are independent gene products, their activities increase in proportion (~20-fold) in the overproducer lines. This strongly suggests that DHFR and TS are not only functionally and physically linked in the same enzyme complex, but also are coregulated. These cell lines resemble the MTX-induced DHFR overproducer amplified cell lines of mammalian origin in their mode of selection, high frequency of appearance, elevated enzyme activity, and increased specific mRNA levels.     

Effects of 4-hydroperoxycyclophosphamide (4-OOH-CP) and 4-hydroperoxydechlorocyclophosphamide (4-OOH-deCICP) on the cell cycle of post implantation rat embryos.

In this study, we used preactivated forms of cyclophosphamide (CP) and dechlorocyclophosphamide (deClCP) to examine the effects of phosphoramide mustard (PM) and acrolein, respectively, on the cell cycle of postimplantation rat embryos. The percentage distribution of cells in the G1/G0, S, and G2/M phases of the cell cycle was determined by flow-cytometric analysis. At embryotoxic concentrations, 4-OOH-CP (PM) induced major cell cycle perturbations whereas 4-OOH-deClCP (acrolein) caused no major perturbation of the cell cycle. These data support the hypothesis that the mechanism of the embryotoxic action of PM involves alkylation of DNA, whereas the mechanism of action of acrolein does not. The primary effect of PM on the cell cycle was an initial delay in the S phase followed by a G2/M arrest. At low embryotoxic concentrations of 4-OOH-CP, there was apparent reversal of the G2/M arrest; at higher embryotoxic concentrations there was little recovery from the G2/M arrest. The high level of cell death found at higher drug concentrations suggests that prolonged G2/M arrest leads to cell death. Using radiolabeled CP and cell sorting, it was determined that PM predominantly alkylated DNA in the S phase of the cell cycle. Overall, the data from this study support the hypothesis that DNA cross-links, induced by the alkylation of DNA by PM, induce cell cycle perturbations. Furthermore, these cell cycle alterations may be one of the early steps in the mechanism leading to the embryotoxicity of PM.     

Gene-specific and strand-specific DNA repair in the G1 and G2 phases of the cell cycle.

We have analyzed the fine structure of DNA repair in Chinese hamster ovary (CHO) cells within the G1 and G2 phases of the cell cycle. Repair of inactive regions of the genome has been suggested to increase in the G2 phase of the cell cycle compared with other phases. However, detailed studies of DNA repair in the G2 phase of the cell cycle have been hampered by technical limitations. We have used a novel synchronization protocol (D. K. Orren, L. N. Petersen, and V. A. Bohr, Mol. Cell. Biol. 15:3722-3730, 1995) which permitted detailed studies of the fine structure of DNA repair in G2. CHO cells were synchronized and UV irradiated in G1 or early G2. The rate and extent of removal of cyclobutane pyrimidine dimers from an inactive region of the genome and from both strands of the actively transcribed dihydrofolate reductase (DHFR) gene were examined within each phase. The repair of the transcribed strand of the DHFR gene was efficient in both G1 and G2, with no major differences between the two cell cycle phases. Neither the nontranscribed strand of the DHFR gene nor an inactive region of the genome was repaired in G1 or G2. CHO cells irradiated early in G2 were more resistant to UV irradiation than cells irradiated in late G1. Since we found no major difference in repair rates in G1 and G2, we suggest that G2 resistance can be attributed to the increased time (G2 and G1) available for repair before cells commit to DNA synthesis.     

Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification

Initiation of DNA synthesis occurs with high frequency at oriß, a region of DNA from the amplified dihydrofolate reductase (DHFR) domain of Chinese hamster CHOC 400 cells that contains an origin of bidirectional DNA replication (OBR). Recently, sequences from DHFR oriß/OBR were shown to stimulate amplification of cis-linked plasmid DNA when transfected into murine cells. To test the role of oriß/OBR in chromosomal gene amplification, linearized plasmids containing these sequences linked to a DHFR expression cassette were introduced into DHFR- CHO DUKX cells. After selection for expression of DHFR, cell lines that contain a single integrated, unrearranged copy of the linearized expression plasmid were identified and exposed to low levels of the folate analog, methotrexate (MTX). Of seven clonal cell lines containing the vector control, three gained resistance to MTX by 5 to 15-fold amplification of the integrated marker gene. Of 16 clonal cell lines that contained oriß/OBR linked to a DHFR mini-gene, only 6 gained resistance to MTX by gene amplification. Hence, sequences from the DHFR origin region that stimulate plasmid DNA amplification do not promote amplification of an integrated marker gene in all chromosomal contexts. In addition to showing that chromosomal position has a strong influence on the frequency of gene amplification, these studies suggest that the mechanism that mediates the experiment of episomal plasmid DNA does not contribute to the early steps of chromosomal gene amplification.     

Vectors containing a prokaryotic dihydrofolate reductase gene transform Drosophila cells to methotrexate-resistance.

Transformed Drosophila Kc cell lines, resistant to methotrexate, an inhibitor of de novo purine and pyrimidine synthesis, have been obtained by calcium phosphate transfection of plasmids containing a sequence coding for a methotrexate-resistant dihydrofolate reductase enzyme (DHFR). The introduced DNA is stably maintained in the cells as head-to-tail multimeric structures of the initial DNA sequence even after several months of culture in the presence of the selective agent. The introduced sequences are present at a high copy number in the transformed cells and express cytoplasmic RNAs transcribed from the DHFR gene.     

Cell proliferation and cell cycle dependent changes in the methylthioadenosine phosphorylase activity in mammalian cells

The objective of this study is to investigate the activity of methylthioadenosine phosphorylase (MTA-Pase) in mammalian cells stimulated by serum to proliferate and during their cell cycle. A direct correlation between growth rate and MTA-Pase activity in chinese hamster ovary (CHO) cells was observed. High MTA-Pase activity was observed during the exponential growth phase followed by a low enzyme activity during plateau phase of growth. To understand whether the fluctuations in the enzyme activity was cell cycle dependent, initially the activity of MTA-Pase was studied in plateau phase (G0) CHO cells as they synchronously go into S phase upon plating in fresh medium. The MTA-Pase activity in G0 cells before initiation of growth was 10.3 n.mol/mg protein/30'. A peak activity of 16.0 n.mol/mg/30 min was found at 12 hr after stimulation of proliferation by serum. These results indicate a peak MTA-Pase activity between 10-12 hr after stimulation of proliferation coinciding with the initiation of DNA synthesis. The activity of the enzyme slowly decreased as the cells completed their DNA synthesis. To understand whether these fluctuations are cell cycle specific, HeLa cells were synchronized in different phases and MTA-Pase activity was studied. The specific activities of the enzyme were 2.76, 2.99, 3.97, 3.28 and 3.65 n.moles/mg/30 min. in mitosis, early G1, late G1, S and G2 phases of the cell cycle respectively. These results indicate that MTA-Pase activity peaks in late G1 phase before the initiation of DNA synthesis, similar to the polyamine biosynthetic enzymes and might play a role in the initiation of DNA synthesis by salvage of adenine into nucleotide pools.     

Initiation of DNA synthesis in the prematurely condensed chromosomes of G1 cells

The objective of this study was to investigate whether G1 cells could enter S phase after premature chromosome condensation resulting from fusion with mitotic cells. HeLa cell synchronized in early G1, mid-G1, late G1, and G2 and human diploid fibroblasts synchronized in G0 and G1 phases were separately fused by use of UV-inactivated Sendai virus with mitotic HeLa cells. After cell fusion and premature chromosome condensation, the fused cells were incubated in culture medium containing Colcemid (0.05 micrograms/ml) and [3H]thymidine ([3H]ThdR) (0.5 microCi/ml; sp act, 6.7 Ci/mM). At 0, 2, 4, and 6 h after fusion, cell samples were taken to determine the initation of DNA synthesis in the prematurely condensed chromosomes (PCC) on the basis of their morphology and labeling index. The results of this study indicate that PCC from G0, G1, and G2 cells reach the maximum degree of compaction or condensation at 2 h after PCC induction. In addition, the G1-PCC from normal and transformed cells initiated DNA synthesis, as indicated by their "pulverized" appearance and incorporation of [3H]ThdR. Further, the initiation of DNA synthesis in G1-PCC occurred significantly earlier than in the mononucleate G1 cells. Neither pulverization nor incorporation of label was observed in the PCC of G0 and G2 cells. These findings suggest that chromosome decondensation, although not controlling the timing of a cell's entry into S phase, is an important step for the initiation of DNA synthesis. These data also suggest that the entry of a S phase may be regulated by cell cycle phase-specific changes in the permeability of the nuclear envelope to the inducers of DNA synthesis present in the cytoplasm.