Mechanism of unbalanced growth-induced cell damage. I. A probable role for hydrolytic enzymes synthesis

This study examines the relationship between cell progress into the state of unbalanced growth, hydrolytic enzyme activities and cell survival during the exposure of L5178Y cells to hydroxyurea (HU), excess thymidine (dThR), hydroxyurea with excess of four deoxyribonucleosides (dNR) or excess dTHR with deoxycytidine (dCR). Cell progress into the state of unbalanced growth was measured as cell size, protein/DNA ratio and protein content per cell. Activities of two lysosomal (acid phosphatase, beta-N-acetylglucosaminidase) and one cytoplasmic non-lysosomal (LDH) enzymes were determined. It has been found that in cells arrested by HU or excess dThR, a progressive cell volume increase with protein/DNA imbalance is correlated with a progressive increase in lysosomal and non-lysosomal hydrolase activities in the cells and in the medium and with a marked lethal effect. Cell volume increase, enhancement of enzyme activities and cell killing could be prevented in HU-arrested cells by concomitant addition of excess dNR (deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine) leading to equal inhibition of DNA and protein synthesis. Control-like values of all parameters were achieved also in cells in which the dThR-inhibiting effect was reversed by dCR addition. It is suggested that a common pathway in the mode of action of the chemotherapeutic agents inducing cell killing through the state of unbalanced growth can be the over-production, abnormal accumulation and progressive leakage of numerous hydrolytic enzymes through the cell membranes, leading in consequence to 'lytic' cell death.     

Relationship between sensitivity to 4'-(9-acridinylamino)methanesulfon-m-anisidide and DNA topoisomerase II in a cold-sensitive cell-cycle mutant of a murine mastocytoma cell line

The cold-sensitive (proliferating at 39.5 degrees C, reversibly arrested in GI-phase at 33 degrees C) cell-cycle mutant 21-Fb of the murine mastocytoma cell line P815 was used to study the effect of amsacrine on non-cycling cells. The sensitivity of arrested 21-Fb cells decreased less than 2-fold in cell survival experiments when compared to proliferating cells. In contrast, DNA breakage and stimulation of protein-DNA complex formation in intact or lysed cells was reduced approx. 10-fold in arrested cells and DNA topoisomerase II activity in arrested cells was only 5% of the activity in proliferating cells. Thus, there was no correlation between cell survival and DNA damage or DNA topoisomerase II activity in drug-treated cells.     

Unbalanced growth and cell death in HeLa S3 cultures treated with DNA synthesis inhibitors

Flow cytometry indicated that significant amounts of dsRNA were accumulated in HeLa S3 cells blocked at or near G₁/S boundary by hydroxyurea (HU) or excess thymidine (TdR). The dsRNA/DNA ratio increased in these cells in a manner characteristic of unbalanced cell growth. In HU-treated cells, dsRNA content was maximal 16 hours after addition of the drug and did not change significantly during the next 24 hours. The DNA content in blocked cells increased by 10%. Cell viability assessed by colony formation in soft agar decreased exponentially in HU-treated cultures after 16 hours of incubation. Correlation between loss of cell viability and rate of cell proliferation after removal of HU was observed, as determined by cell count and analysis of cell cycle progression. In TdR-treated cultures cells slowly progressed into mid S-phase during 40 hours and dsRNA accumulation continued during this period. Cell viability was not significantly affected by treatment with excess TdR, indicating that unbalanced growth per se, as measured by dsRNA accumulation, is not lethal for the cells. After reversal of DNA synthesis inhibition by removal of the drug, cells treated with HU for 16 hours or TdR for 16–24 hours promptly progressed through the cell cycle. This progression was accompanied by accumulation of significant amounts of dsRNA. As a result, cells in G₂ phase had a very high dsRNA content leading to retention of the unbalanced condition (increased dsRNA/DNA ratio) in the daughter cells. It is suggested that dsRNA accumulation in the cell is controlled to a certain degree by cell progression through the S phase. This type of control, evidently, was reflected in limited dsRNA accumulation in the cells blocked at or near G₁/S border, in continuous dsRNA accumulation in the cells slowly progressing through S phase, and in accumulation of large amounts of dsRNA after renewal of progression through the S phase.     

ERK1/2 mediates unbalanced growth leading to senescence induced by excess thymidine in human cells

Excess thymidine induces unbalanced growth by delaying DNA replication and subsequently induces senescence in every human cell type. Our previous studies with use of inhibitors suggested that ERK1/2 has a major role in these processes. Here we directly assessed the roles of ERK1 and ERK2 in unbalanced growth induced by excess thymidine. Knockdown of ERK2 and ERK1 by vector-based RNA interference prevented loss of colony forming ability and appearance of senescence markers induced by excess thymidine in HeLa and TIG-7 cells, respectively. Such cells continued growing in the presence of excess thymidine. Double knockdown of ERK1 and ERK2 did not improve the effects of single knockdowns of ERK1 and ERK2 in either cell types. These results demonstrate that ERK1 or ERK2 has a major role in manifestation of unbalanced growth in human cells.     

DNA damage and repair in relation to cell killing in neocarzinostatin-treated HeLa cells.

To elucidate the mechanism of the cell killing activity of neocarzinostatin on mammalian cells, the drug-induced damage of DNA and its repair were examined. Very low doses of neocarzinostatin, at which high survival of cells was observed, clearly produced single-strand breaks of DNA and decomposition of the 'DNA complex', but these damages appeared to be repaired almost completely. At higher doses of neocarzinostatin, single-strand breaks were repaired to a considerable extent while double-strand breaks seemed not to be repaired. The number of non-repairable single-strand breaks was about twice that of double-strand breaks. This implies that single-strand breaks are repaired except for those constituting double-strand breaks. Although at low levels of neocarzinostatin repair of double-strand breaks may occur, the correlation existing between the colony-forming ability of cells treated with neocarzinostatin and non-repairable DNA breakage suggests that production of a small number of critical non-repairable double-strand breaks per cell may be responsible for the cell killing activity of the drug.     

ADP-ribosyltransferase activity during the friend virus-induced murine erythroleukemia cell differentiation

A variety of chemical agents that are known to induce erythrodifferentiation in the Friend virus-induced murine erythroleukemia (MEL) cell have been suggested to mediate DNA cleavage in cultured cells prior to differentiation. The activation of the nuclear enzyme, ADP-ribosyltransferase, depends upon the presence of single strand breaks in DNA. If dimethyl sulfoxide (Me2SO) causes DNA breakage, it would be expected that the activity of ADP-ribosyltransferase would increase. A study of ADP-ribosyltransferase activity during cell growth indicates that both Me2SO-treated and untreated MEL cells exhibit a similar increase in the enzyme activity but the increase in Me2SO-treated cells is delayed by a few hours. When examined at comparable stages of growth, both treated and untreated cells show almost identical levels of enzyme activity. The present data thus do not support the contention that Me2SO induces DNA breakage in the MEL cells.     

Yeast-phase cell cycle of the polymorphic fungus Wangiella dermatitidis.

The yeast-phase cell cycle of Wangiella dermatitidis was studied using flow microfluorimetry and the deoxyribonucleic acid (DNA) synthesis inhibitor hydroxyurea (HU). Exposure of exponential-phase yeastlike cells to 0.1 M HU for 3 to 6 h resulted in the arrest of the cells in DNA synthesis and produced a nearly homogeneous population of unbudded cells. Treatment of the yeast-phase cells with HU for 9 h or longer resulted in the accumulation of the cells predominantly as budded forms having either a single nucleus in the mother cell or a single nucleus arrested in the isthmus between the mother cell and the daughter bud. Exposure of unbudded stationary-phase cells to 0.1 M HU resulted in the accumulation of the cells in the same phenotypes. Analysis by flow microfluorimetry and cell counts of HU-inhibited mithramycin-stained cells indicated that the eventual progress of HU-inhibited cells from unbudded to the two budded forms was due to the limited continuation of the growth sequence of the cell cycle even in the absence of DNA synthesis, nuclear division, and in some cases nuclear migration. On the basis of these observations and the results of flow microfluorimetric analysis of exponential-phase cells, a map of the yeast-phase cell cycle was constructed. The cycle appears to consist of two independent sequences of events, a budding growth sequence and a DNA division sequence. The nuclear division cycle of yeast-phase cells growing exponentially with a 4.5-h generation time is composed of a G1 interval of 148 min, as S phase of 16 min, and a G2 plus M interval of 107 min.     

Assessment of cell viability by flow cytometric analysis using DNase exclusion

A new method was developed for selective measurement of DNA distributions in viable cell populations. The method is based on the fact that non-viable cells lose membrane integrity and treatment of such cells with DNase should remove their DNA. The DNase-treated cells were stained with DNA fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) in the presence of Triton X-100. DNA distribution was measured by flow cytometry prior to and after treatment with DNase. Percentage of cells stained after DNase treatment was considered as an index of cell viability. Optimal conditions for DNase treatment and application of DNase exclusion test for the analysis of spontaneous cell death, selective death of cells arrested in S/G2 phases, instant cell disintegration induced by cytotoxic compounds and cell death induced by hyperthermia are described.     

DNA damage induced by bleomycin in the presence of dibucaine is not predictive of cell growth inhibition

Growth inhibition and cell killing by bleomycin are believed to be related to the ability of this antibiotic to cleave chromosomal DNA. Because bleomycin has an intracellular site of action, its ability to cross biological membranes must be critical to its overall effectiveness as an antitumor agent. The local anesthetic dibucaine acts to enhance membrane fluidity; therefore, the reported ability of this local anesthetic to modulate bleomycin effects on KB cells was investigated. Cells were treated with various bleomycin congeners in the presence or absence of dibucaine for 24 h. Dibucaine enhanced the inhibition of cell growth mediated by bleomycin A2, demethylbleomycin A2, bleomycin B2, and isobleomycin A2. N-Acetylbleomycin A2 did not inhibit cell growth in the absence of dibucaine, but it was inhibitory in the presence of dibucaine. Cells treated simultaneously for analysis of DNA breakage on alkaline sucrose gradients revealed that breakage was also enhanced in the presence of dibucaine. The degree of enhancement varied with dose and bleomycin congener. N-Acetylbleomycin A2 did not induce DNA breakage in either the absence or the presence of dibucaine. While growth inhibition and net DNA breakage correlated reasonably well in the absence of dibucaine for each bleomycin analogue tested, proportionality was lost in the presence of dibucaine, and very little DNA breakage was present when growth inhibition was complete. These observations imply that, at least in the presence of dibucaine, bleomycin may mediate growth inhibition at some locus in addition to chromosomal DNA and, also, that a given net amount of bleomycin analogue induced DNA damage per se does not produce a specific degree of growth inhibition.     

Deoxyribonuclease II activity in relation to cell cycle in synchronized HeLa S3 cells

Studying the activity of DNase II in relation to cell cycle in synchronized HeLa S3 cells show a two to seven fold increase in DNase II activity at those times when DNA synthesis is taking place. The peaks of DNase II activity coincide with the peaks of DNA synthesis. The increased DNase II activity could be prevented by puromycin, suggesting that the enzyme activity increased at the S phase was caused by synthesis of new molecules rather than the activation of existing molecules. Acid phosphatase (as a marker for lysosomal enzymes) does not show an induction similar to that observed for DNase II in relation to cell cycle.     

The influence of oxygen on the survival and yield of DNA double-strand breaks in irradiated yeast cells.

Survival and induction of DNA double-strand breaks were studied in cells of Saccharomyces cerevisiae irradiated under oxic or anoxic conditions with 30 MeV electrons. A linear relationship between DNA double-strand breakage and dose was found in both cases. The o.e.r.-value for colony forming ability was found to be 1.9 +/- 0.2, whereas the o.e.r.-value for DNA double-strand breakage was 3.0 +/- 0.1. These results are not inconsistent with the idea that DNA double-strand breaks are involved in killing of yeast cells. The frequency of induction of DNA double-strand breaks was found to be 0.74 x 10(-11) double-strand breaks per g/mol per Gy when cells were irradiated under oxygen and 0.24 x 10(-11) double-strand breaks per g/mol per Gy under nitrogen.     

Topoisomerase-specific drug sensitivity in relation to cell cycle progression.

The nuclear enzyme DNA topoisomerase II catalyzes the breakage and resealing of duplex DNA and plays an important role in several genetic processes. It also mediates the DNA cleavage activity and cytotoxicity of clinically important anticancer agents such as etoposide. We have examined the activity of topoisomerase II during the first cell cycle of quiescent BALB/c 3T3 cells following serum stimulation. Etoposide-mediated DNA break frequency in vivo was used as a parameter of topoisomerase II activity, and enzyme content was assayed by immunoblotting. Density-arrested A31 cells exhibited a much lower sensitivity to the effects of etoposide than did actively proliferating cells. Upon serum stimulation of the quiescent cells, however, there was a marked increase in drug sensitivity which began during S phase and reached its peak just before mitosis. Maximal drug sensitivity during this period was 2.5 times greater than that of log-phase cells. This increase in drug sensitivity was associated with an increase in intracellular topoisomerase II content as determined by immunoblotting. The induction of topoisomerase II-mediated drug sensitivity was aborted within 1 h of exposure of cells to the protein synthesis inhibitor cycloheximide, but the DNA synthesis inhibitor aphidicolin had no effect. In contrast to the sensitivity of cells to drug-induced DNA cleavage, maximal cytotoxicity occurred during S phase. A 3-h exposure to cycloheximide before etoposide treatment resulted in nearly complete loss of cytotoxicity. Our findings indicate that topoisomerase II activity fluctuates with cell cycle progression, with peak activity occurring during the G2 phase. This increase in topoisomerase II is protein synthesis dependent and may reflect a high rate of enzyme turnover. The dissociation between maximal drug-induced DNA cleavage and cytotoxicity indicates that the topoisomerase-mediated DNA breaks may be necessary but are not sufficient for cytotoxicity and that the other factors which are particularly expressed during S phase may be important as well.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

Stimulation of intracellular topoisomerase I activity by vasopressin and thrombin. Differential regulation by pertussis toxin.

Incubation of cultured rat aortic smooth muscle cells (A-10, ATCC CRL 1476) with [8-arginine]vasopressin (AVP) or thrombin increased the amount of DNA strand breakage induced by camptothecin, an inhibitor of topoisomerase I (DNA topoisomerase; EC 5.99.1.2) and transiently stimulated the extractable activity of this enzyme. Both topoisomerase-related responses were prevented by treatment of the cells with AVP or thrombin plus the appropriate receptor antagonist. The increase in strand breakage mediated by AVP and thrombin depended on the concentration of hormone. Neither AVP nor thrombin had any effect on strand breaks obtained with the epipodophyllotoxin VM-26, an inhibitor of topoisomerase II [DNA topoisomerase (ATP-hydrolysing); EC 5.99.1.3]. Pretreatment of the cells with pertussis toxin partially inhibited thrombin-mediated increases in camptothecin-induced strand breakage whereas AVP-mediated increases were unaffected. These results are consistent with the notion that AVP and thrombin induce a transient increase in intracellular topoisomerase I activity via interactions with their respective cell surface receptors and that the effects of the activation of these receptors are mediated by different G-proteins.     

Sensitization of Escherichia coli C to gamma-radiation by 5-bromouracil incorporation.

Escherichia coli C cells, unifilarly substituted with 5-bromouracil (BrUra) were 2-25 times as sensitive as unsubstituted cells to killing by gamma-irradiation under aerobic conditions. The yield of DNA double-strand breaks in BrUra-substituted cells was increased by a factor only 1-55, suggesting that other lesions also contribute to cell-killing. Alkaline sucrose density gradient analysis of the 3H-thymine labelled DNA strand showed there was less repair of gamma-ray-induced single-strand breaks when BrUra was in the complementary strand. Since there are more of these unrepaired breaks than can be accounted for by BrUra-induced DNA double-strand breakage, some fraction of the lethal events in BrUra-substituted E. coli cells may be unrepaired DNA single-strand breaks.     

Single-strand breakage on binding of DNA to cells in the genetic transformation of Diplococcus pneumoniae.

Mutants of Diplococcus pneumoniae that lack a membrane-localized DNAase are defective in transformation because entry of DNA into the cell is blocked. Such mutants still bind DNA on the outside of the cell. The bound DNA is double-stranded and its double-stranded molecular weight is unchanged. Its sedimentation behavior in alkali, however, shows that it has undergone single-strand breakage. The breaks are located randomly in both strands of the bound DNA at a mean separation of 2 × 10⁶ daltons of single-stranded DNA. Both binding and single-strand breakage occur in the presence of EDTA. Single-strand breaks are similarly formed on binding of DNA to normally transformable cells in the presence of EDTA. The single-strand breaks appear to be a consequence of attachment. DNA may be bound to the cell surface at the point of breakage.A mutant that is partially blocked in entry also binds DNA mainly on the outside of the cell. In the presence of EDTA, DNA bound by this mutant undergoes only single-strand breaks. In the absence of EDTA, however, double-strand breaks occur, apparently as a result of the initiation of entry. It is possible that the double-strand breaks arise from additional single-strand breaks opposite those that occurred on binding. The double-strand breaks presumably result from action of the membrane DNAase as it begins to release oligonucleotides from one strand segment while drawing the complementary strand segment into the cell.     

Topoisomerase II activity in a DNA double-strand break repair deficient Chinese hamster ovary cell line

Topoisomerase II activity was measured in wild-type, Chinese hamster ovary K1 cells, and in the DNA double-strand break repair deficient xrs-6 cell line. Total topoisomerase II activity in a high salt, nuclear extract was found to be the same in both cell lines, as measured by decatenation of kinetoplast DNA networks and catenation of plasmid pBR322 DNA. While at low drug concentrations m-AMSA-induced enzyme cutting of nuclear DNA was 25% less in xrs-6 cells, the frequency of DNA breaks at high concentrations of the drug, and thus the frequency of the topoisomerase II enzyme, was the same in both cell lines. Despite the presence of equivalent enzyme levels in both cell lines, the xrs-6 cell line was 3 times more sensitive to drug-induced cytotoxicity. These results may be due to the fact that, as with X-radiation-induced DNA damage, xrs-6 cells are deficient in the capacity to rejoin topoisomerase II-induced DNA double-strand breaks.     

Potentiation of DNA damage by inhibition of poly(ADP-ribosyl)ation: a test of the hypothesis for random nuclease action.

Poly(ADP-ribosyl)ation is a cellular response to DNA strand breaks by which a large array of proteins becomes covalently modified for a brief period during the lifetime of the DNA breaks. Inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide after many types of DNA damage leads to a marked increase in DNA strand breakage, repair replication, cytogenetic damage, mutagenesis, and cell killing. It has been hypothesized that poly(ADP-ribose) polymerase may modify potentially degradative endogenous nucleases that can reduce cellular viability. Thus, in the presence of DNA strand breakage, the polymer would bind these enzymes to inhibit their activity. When synthesis of the polymerase is inhibited, the enzymes would act randomly to produce nonspecific damage in the DNA. We tested this hypothesis by electroporating restriction enzymes into human cells containing the shuttle vector pHAZE. Restriction enzymes cleave at specific recognition sequences in the lacZ target gene of pHAZE, and mutations result from rejoining errors at the cleavage sites. If the hypothesis were correct, enzyme-treated cells cultured with 3-aminobenzamide to inhibit synthesis of poly(ADP-ribose) polymers would result in a significant increase in mutations outside the restriction enzyme sites. The spectrum of mutations observed after electroporation of PvuII (which produces blunt-end double-strand breaks) or PvuI (which produces cohesive-end double-strand breaks) was similar in untreated and 3-aminobenzamide-treated cells. Thus, our results do not support the hypothesis that the increase in damage observed when poly(ADP-ribosyl)ation is inhibited is due to a chaotic, nonspecific attack on DNA by endogenous cellular nucleases.     

Role of deoxyribonucleic acid polymerase III in the repair of single-strand breaks produced in Escherichia coli deoxyribonucleic acid by gamma radiation.

Cell survival, deoxyribonucleic acid (DNA) degradation, and the repair of DNA single-strand breaks were measured for Escherichia coli K-12 pol+, polA1, polC1026(ts), and polA1 polC1026(ts) cells after 137Cs gamma irradiation. The results indicate that DNA polymerase III is required for growth medium-dependent (type III) repair in polA+ or polA cells. In pol+ or polC cells, DNA polymerase I performs type II repair efficiently. The relative deficiencies of each of these strains in DNA repair generally correlate with their relative sensitivities to cell killing and with the extent of DNA degradation observed.