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.     

The cell cycle-coupled expression of topoisomerase IIalpha during S phase is regulated by mRNA stability and is disrupted by heat shock or ionizing radiation.

Topoisomerase II is a multifunctional protein required during DNA replication, chromosome disjunction at mitosis, and other DNA-related activities by virtue of its ability to alter DNA supercoiling. The enzyme is encoded by two similar but nonidentical genes: the topoisomerase IIalpha and IIbeta genes. In HeLa cells synchronized by mitotic shake-off, topoisomeraseII alpha mRNA levels were found to vary as a function of cell cycle position, being 15-fold higher in late S phase (14 to 18 h postmitosis) than during G1 phase. Also detected was a corresponding increase in topoisomerase IIalpha protein synthesis at 14 to 18 h postmitosis which resulted in significantly higher accumulation of the protein during S and G2 phases. Topoisomerase IIalpha expression was not dependent on DNA synthesis during S phase, which could be inhibited without effect on the timing or level of mRNA expression. Mechanistically, topoisomerase IIalpha expression appears to be coupled to cell cycle position mainly through associated changes in mRNA stability. When cells are in S phase and mRNA levels are maximal, the half-life of topoisomerase IIalpha mRNA was determined to be approximately 30 min. A similar decrease in mRNA stability was also induced by two external factors known to delay cell cycle progression. Treatment of S-phase cells, at the time of maximum topoisomerase IIalpha mRNA stability, with either ionizing radiation (5 Gy) or heat shock (45 degrees C for 15 min) caused the accumulated topoisomerase IIalpha mRNA to decay. This finding suggests a potential relationship between stress-induced decreases in topoisomerase IIalpha expression and cell cycle progression delays in late S/G2.     

Cell cycle-specific activity of type I and type II cyclic adenosine 3':5'-monophosphate-dependent protein kinases in Chinese hamster ovary cells.

Types I and II cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases have been studied during the cell cycle of Chinese hamster ovary cells. Chinese hamster ovary cells were synchronized by selective detachment of mitotic cells from monolayer cultures. Protein kinases were separated by DEAE-cellulose chromatography and were similar to the types of cAMP-dependent protein kinases studied in skeletal muscle and in heart extracts. The total amount of protein kinases activity per cell was substantial, both in mitosis and at the G1/S boundary. During mitosis, the relatively high activity of protein kinase was due to a predominance of type I protein kinase. During early G1, the activity of type I protein kinase decreased and there was little detectable type II activity. A rapid increase in the activity of type II was evident at the G1/S boundary. The administration of puromycin (50 mug/ml) from 1 to 5 hours after selective detachment of mitotic cells abolished the activity of type II cAMP-dependent protein kinase seen at the G1/S border, but had no observable effect on the activity of type I protein kinase. The data presented demonstrate cell cycle-specific activity patterns of type I and type II protein kinase Type I protein kinase activity is high in mitosis and is constant throughout the cell cycle. Increased type II protein kinase activity seems to be related to the initiation of DNA synthesis in S phase. The data suggest a translational control of type II cAMP-dependent protein kinase activity.     

The concentration-dependent diversity of effects of DNA topoisomerase I and II inhibitors on the cell cycle of HL-60 cells.

Exposure of promyelocytic leukemic HL-60 cells to 3-60 nM of the DNA topoisomerase I inhibitor camptothecin (CAM) or to 30-450 nM and 0.12-1.5 microM of DNA topoisomerase II inhibitors teniposide (TN) and 4-(9-acridynylamino)-3-methanesulfon-m-anisidide (m-AMSA), respectively, resulted in two distinct kinetic effects: (1) the cells entered S phase but the rate of DNA replication was reduced in proportion to the inhibitor concentration; (2) the transition from G2 to M was impaired, approximately 1 h after addition of the inhibitor. As a consequence, the cells accumulated in the S (preferentially in early S) and in G2 phases of the cell cycle. Whereas CAM was more efficient in suppressing cell progression through S phase, TN and m-AMSA were more potent G2 blockers. At these low inhibitor concentrations no signs of immediate cytotoxicity or DNA degradation were apparent. However, above 145 nM of CAM, 900 nM of TN, or 2 microM of m-AMSA extensive DNA degradation in nuclei of S phase cells was evident within 6 h of addition of the inhibitor, resulting in the loss of S and G2 + M cells from these cultures. The data indicate that depending on concentration, mechanisms mediating the cytostatic/cytotoxic activity of both DNA topoisomerase I and II inhibitors may be quite different. Suppression of the DNA replication and the G2 to M transition, seen at low inhibitor concentrations, is compatible with the assumption that the inhibitor-induced stabilization of the topoisomerase-DNA cleavable complexes interferes with DNA replication and chromosome condensation/segregation, respectively. Above the threshold concentration for each inhibitor, an endonucleolytic activity is triggered, resulting in rapid DNA degradation in nuclei of S and G2 phase cells. The endonucleolytic effect is not only cell cycle phase-specific but is also modulated by tissue-specific factors because it cannot be observed, e.g., in the lymphocytic leukemic cell lines.     

Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells.

The cellular content of 170kD and 180kD topoisomerase II was studied as a function of the proliferation state and cell cycle position in NIH-3T3 cells. When the cells were synchronized by serum starvation and then stimulated to enter the cell cycle by addition of fresh growth medium, the amount of 170kD topoisomerase II present was undetectable until the cells reached late S phase, peaked in G2-M phase cells, and decreased as the cells completed mitosis. The amount of 180kD topoisomerase II was constant once the cells entered the cell cycle. When exponentially growing cells were induced to enter G0 by serum starvation, the amount of 170kD topoisomerase II decreased in parallel with the loss of cells from the S and G2-M phases of the cell cycle and was undetectable once all of the cells reached G0. In contrast, the 180kD enzyme was still present after all of the cells had entered G0. The tightness of association of the two enzymes with chromatin was measured by determining the concentration of salt required to extract them from isolated nuclei. The 180kD enzyme required a higher concentration of NaCl for extraction than did the 170kD enzyme. The different patterns of expression of the two forms of topoisomerase II suggest that they perform different functions in cells.     

Productive HIV-1 infection of macrophages restricted to the cell fraction with proliferative capacity.

Retroviruses establish productive infection only in proliferating cells. Macrophages are often considered to be non-proliferating in vitro yet are susceptible to HIV-1 infection. This has led to the conclusion that HIV-1 can establish infection independent of host cell proliferation. We here report that a small proportion of macrophages does have proliferative capacity. A comparable small fraction of monocyte derived macrophages (MDM) supported productive HIV-1 infection as demonstrated in limiting dilution culture. Fluorescence activated cell sorting on the basis of incorporation of BrdUrd, a thymidine analog, and subsequent PCR analysis revealed the presence of proviral DNA only in the BrdUrd positive cell fraction with DNA synthesizing activity. To identify which phase of cell cycle is required for establishment of productive infection, growth arrest in G1 or G1/S phase prior to inoculation was performed. gamma-Irradiation, which arrests primary cells in G1, prevented both cell proliferation and establishment of productive infection in MDM. Treatment of MDM with aphidicolin, a specific inhibitor of DNA polymerase alpha and delta which arrests cells in G1/S phase of the cell cycle, also inhibited DNA synthesis but did not prevent establishment of productive infection which is completely analogous to observations in T cells. Our data thus indicate that not cell division itself but cellular conditions that coincide with cell proliferation are apparently indispensable for establishment of productive infection.     

Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes.

The mammalian homologue of the cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a p34cdc2 cyclin-dependent kinase that regulates the cell cycle of a wide variety of cell types. Resting murine T lymphocytes contained no detectable p34cdc2 protein, histone kinase activity, or specific mRNA for the cdc2 gene. Activation of the T cells by immobilized anti-CD3 resulted in the expression of specific mRNA late in the G1 phase of the cell cycle, and p34cdc2 protein was detectable at or near G1/S. At this point in the cell cycle, the protein was phosphorylated at tyrosine and displayed no H1 histone kinase activity. As the cells progressed through the cycle, the amount of specific mRNA and p34cdc2 increased, and H1 histone kinase activity was detectable when the cells were blocked at G2/M by nocodazole. The activation of T cells by phorbol dibutyrate induced the expression of IL-2R but failed to induce the synthesis of IL-2 or the expression of cdc2-specific mRNA. Under these conditions, the activated cells failed to enter the S phase of the cell cycle. Because the presence of IL-2 added exogenously during activation by phorbol dibutyrate resulted in the expression of cdc2-specific mRNA and progression through the cell cycle, either IL-2 or the interaction with IL-2R may be involved in the expression of cdc2 and regulation of the G1/S transition.     

The G1 interval in the mammalian cell cycle: dual control by mass accumulation and stage-specific activities

The temporal determinants of the G1 cell cycle interval were investigated using nine mammalian cell lines. In each case, cells were allowed to proliferate for many cell cycles under conditions that slowed progress through S phase without an equivalent impairment of overall mass accumulation. This disproportionate inhibition of progress through the cell cycle caused newly produced cells to be more massive than usual. Under these growth conditions, the determinants of the length of the G1 interval became evident. For two cell lines, HeLa S3 and NIH 3T3, a protracted S phase, and the resultant increase in mass, resulted in a dramatically shortened G1 interval. Thus, for these cell lines, a major portion of G1 time exists to accommodate mass accumulation needed to initiate the subsequent S phase. Nevertheless, under conditions that protracted S phase and shortened the G1 interval, cells still exhibited a measurable G1 time, reflecting the stage-specific activities within G1. One activity that may be responsible for this obligatory G1 time is the synthesis of a labile protein. For other cells studied here, protraction of S phase also caused proliferating cells to become more massive, but in these cases there was no diminution of the G1 time. For these cells, the entire G1 interval must accommodate G1-specific activities necessary to initiate a new cell cycle. A unifying view of the G1 interval recognizes the two distinct influences that determine the time spent in G1: the need to accumulate sufficient mass to initiate a new DNA-division sequence; and the stage-specific events necessary for the subsequent S phase. The length of the G1 interval is dictated by the longer of these two time-consuming activities.     

Regenerative proliferation of mouse epidermal cells following adhesive tape stripping. Micro-flow fluorometry of isolated epidermal basal cells combined with 3H-TdR incorporation and a stathmokinetic method (colcemid).

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) Laerum, 1969) and brought into a monodisperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G1, S and (G1 + M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. 3H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the G1 phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

Cell cycle stage dependent variations in drug-induced topoisomerase II mediated DNA cleavage and cytotoxicity

The DNA cleavage produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in mammalian cells is putatively mediated by topoisomerase II. We found that in synchronized HeLa cells the frequency of such cleavage was 4-15-fold greater in mitosis than in S while the DNA of G1 and G2 cells exhibited an intermediate susceptibility to cleavage. The hypersensitivity of mitotic DNA to m-AMSA-induced cleavage was acquired relatively abruptly in late G2 and was lost similarly abruptly in early G1. The susceptibility of mitotic cells to m-AMSA-induced DNA cleavage was not clearly paralleled by an increase in topoisomerase II activity (decatenation of kinetoplast DNA) in 350 mM NaCl extracts from mitotic cells compared to similar extracts from cells in G1, S, or G2. Furthermore, equal amounts of decatenating activity from cells in mitosis and S produced equal amounts of m-AMSA-induced cleavage of simian virus 40 (SV40) DNA; i.e., the interaction between m-AMSA and extractable enzyme was similar in mitosis and S. The DNA of mitotic cells was also hypersensitive to cleavage by 4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (etoposide), a drug that produces topoisomerase II mediated DNA cleavage without binding to DNA. Thus, alterations in the drug-chromatin interaction during the cell cycle seem an unlikely explanation for results in whole cells. Cell cycle stage dependent fluctuations in m-AMSA-induced DNA cleavage may result from fluctuations in the structure of chromatin per se that occur during the cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoplasmic and nuclear protein kinases during the cell cycle.

Nuclear and cytoplasmic protein kinases were measured during the traverse of synchronous CHO cultures through G1 into S phase. Cells were synchronized by selective detachment of cells blocked in metaphase using colcemid. Nuclei were isolated and the protein kinases extracted from the nuclear preparation with 0.6 M NaCl. This procedure solubilized greater than 90% of the total protein kinase activity present in the nuclear preparation. DEAE chromatography of this extract showed 5 apparently different ionic forms of nuclear protein kinases. The nuclear protein kinases preferred casein and phosvitin to histone as substrates and were cyclic AMP-independent. Nuclear protein kinase activities increased greater than two-fold, when expressed as units of activity per cell nucleus, during G1 phase traverse, concomitant with a 70% increase in nuclear non-histone proteins (those soluble in 0.6 M NaCl). This resulted in only a 40% increase in the specific activities (units/microgram protein in 0.6 M NaCl extractable nuclear fraction) of these enzymes as cells progressed through G1 into S phase. This was in contrast to cytoplasmic cyclic AMP-dependent protein kinase activities which also increased two-fold during progression through G1 phase while total cellular protein increased less than 20%. Activation of, as well as synthesis of, cyclic AMP-dependent cytoplasmic protein kinases during G1 phase suggests a regulatory mechanism for precise temporal phosphorylation, whereas the constant specific activity in nuclear kinases during cell cycle is more compatible with the maintenance of bulk phosphorylation processes in the nucleus.     

Ras-dependent cell cycle commitment during G2 phase

Synchronization used to study cell cycle progression may change the characteristics of rapidly proliferating cells. By combining time-lapse, quantitative fluorescent microscopy and microinjection, we have established a method to analyze the cell cycle progression of individual cells without synchronization. This new approach revealed that rapidly growing NIH3T3 cells make a Ras-dependent commitment for completion of the next cell cycle while they are in G2 phase of the preceding cell cycle. Thus, Ras activity during G2 phase induces cyclin D1 expression. This expression continues through the next G1 phase even in the absence of Ras activity, and drives cells into S phase.     

A new action for topoisomerase inhibitors.

Topoisomerases are known to aid DNA replication by breaking and resealing supercoiled DNA. Consequently, cells exposed to topoisomerase inhibitors before or during the S (DNA synthetic) phase of the cell cycle undergo abnormal DNA replication and become irreversibly blocked in the G2 (pre-mitosis) phase. We report that following a 4-h exposure to topoisomerase II inhibitors, murine erythroleukemic cells (MELC) do not form mitotic figures but exhibit a time-dependent progression into G2 (4N DNA) and greater than G2 (up to 8N DNA) stages of the cell cycle. Following exposure to the topoisomerase I inhibitor camptothecin, recovering MELC also exhibit greater than G2 polyploidy, but to a considerably lesser degree: mitotic figures are present and a subpopulation of cells resumes cycling. However, both topo I and topo II inhibitors induce maximal percentages of greater than G2 cells when synchronized MELC are in the G2/M phase at the time of exposure. This suggests that, in addition to their S-phase action, topoisomerase inhibitors can interfere with chromosome condensation during G2 and, in so doing, induce polyploidy.     

Functional interactions of DNA topoisomerases with a human replication origin

The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.     

T98G glioma cells have nicks in DNA in quiescent phase

Human glioblastoma-derived cell line, T98G, is arrested in the G1 phase of the cell cycle when serum is deprived. Using this cell line, we investigated the relation between the cell cycle and DNA single-stranded breaks, "nicks," by an in situ nick-translation method. When T98G cells were cultured without serum for 60 h, many small cells with condensed chromatin and scanty cytoplasm appeared. These small cells that were immunohistochemically considered to be in the G0 or early G1 phase had many nicks in DNA. When serum was added, these small cells with nicks disappeared within 1 to 4 h. VP-16, a DNA topoisomerase II inhibitor, delayed the disappearance of these small cells with nicks. This indicated that the action of DNA topoisomerase II on the chromatin is required to repair nicks in T98G glioma cells and to promote the progression from the quiescent to the proliferating phase.     

Impaired S-phase transit of Werner syndrome cells expressed in lymphoblastoid cell lines

The clinical phenotype of Werner's syndrome (WS) includes short stature, premature cataracts, skin atrophy, osteoporosis, graying and loss of hair, neoplasia, diabetes mellitus, and arteriosclerosis. Cultured cells from patients with this autosomal recessive disorder exhibit chromosomal instability and a markedly reduced replicative lifespan and growth rate. To elucidate the cell cycle alterations associated with the growth deficit, we continuously labeled lymphoid cell lines from five WS patients and from four healthy adult controls with 5-bromodeoxyuridine. Bivariate Hoechst 33258/ethidium bromide flow cytometry revealed a 2.4-h prolongation in the minimal duration of the S phase of WS cells (P less than 0.005). Moreover, the fraction of proliferating cells irreversibly arrested in the S phase (5.4% vs 1.4% in controls) was significantly elevated in WS (P less than 0.001). Other cell cycle compartments were not significantly affected in WS cell lines. As a partial test of the hypothesis that the WS phenotype is due to a defect in DNA topoisomerase I (topo I) or DNA topoisomerase II (topo II) we exposed lymphoid cells from a healthy control to the topo I inhibitor camptothecin or to the topo II inhibitor 4'-(9-acridinylamino)methanesulfon-m-anisidine. The cell kinetic alterations elicited by these compounds differed from that exhibited by untreated WS patients. Thus, a primary defect in topo I or II is unlikely in WS. Our cell cycle results, however, provide important evidence that the biochemical genetic lesion is in fact expressed in lymphoblastoid cell lines, the most readily available cells from such subjects.