Chromosomes and DNA-replication of rat kangaroo cells (PtK2)

The karyotype, chromosomal measurements, and the time course of DNA replication during the S-phase were determined in metaphase chromosomes of non-synchronized monolayer cultures of PtK₂ cells (CCL 56) derived from Potorous tridactylis. The karotype was the same as originally determined for this cell line. Chromosomal measurements differed from data for primary bone marrow cells of this species published by Shaw and Krooth. PtK₂ cells and chromosomes showed maximal incorporation of tritiated thymidine (³H-TdR) halfway through the S-phase. Chromosome Y₁ showed a second peak of ³H-TdR-incorporation at the end of the S-phase in addition to the peak halfway through S. Comparison of grain densities for chromosomal arms showed late replication of the short arms of chromosomes 1, 3, and X. The time course of incorporation of ³H-TdR was changed when cells were treated for 1 h with fluorodeoxyuridine (FUdR) prior to the ³H-TdR-pulse. FUdR-treated cells showed maximum incorporation of ³H-TdR immediately after the beginning of the S-phase, which was followed by a second peak halfway through the S-phase. This indicated that ³H-TdR-incorporation was partially synchronized by treatment of cells with FUdR. Total radioactivity of FUdR-treated cells had increased by 77% in comparison to cells not treated with FUdR, which indicates that approximately 44% of the TdR-precursors of the latter cells may have originated from cellular precursor pools.     

INCORPORATION OF TRITIATED THYMIDINE BY EUCARYOTIC MICROALGAE1

The uptake and incorporation of tritiated thymidine (³H-TdR) by axenic laboratory cultures of marine diatoms and dinoflagellates was measured. ³H-TdR was incorporated into nucleic acids by all four algae examined during a two to six hour period prior to cytokinesis and not during other times of the cell cycle. Between 90-95% of the ³H label incorporated into (cold trichloroacetic acid insoluble) nucleic acids was recovered from DNA. Incorporation of ³H-TdR appears to accurately indicate the timing of DNA synthesis. The incorporation of ³H-TdR by eucaryotic algae during long term (24 h) incubations does not generally preclude using ³H-TdR uptake to estimate bacterial production and growth during short term incubations.     

Cell cycle kinetics, tissue transglutaminase and programmed cell death (apoptosis).

Studies were undertaken on a highly metastatic hamster fibrosarcoma cell line with a view to assessing whether cells entering into apoptosis, measured by counting the number of transglutaminase mediated detergent insoluble envelopes, has any synchrony with a particular phase of the cell cycle. A double exposure of thymidine was used to block cells in early S-phase. Flow cytometry in combination with [3H]thymidine incorporation into DNA was used to assess the degree of synchrony and progression through the different phases of cell cycle. The apoptotic index was found to be at its maximum in mid-S-phase. Measurement of transglutaminase activity in each phase of the cell cycle indicated that the specific activity was also at its greatest during mid S-phase. The level of enzyme was relatively unchanged throughout the cell cycle indicating that the regulation of transglutaminase activity occurs primarily through effects on catalytic activity rather than enzyme synthesis.     

Difference between diploid and aneuploid Chinese hamster cells in replication at mid-S-phase

Following partial synchronization of the heteroploid Chinese hamster cell line V-79 and of normal diploid lung fibroblasts of the Chinese hamster in culture, their DNA replication during S-phase was compared by means of a BrdU-incorporation/thymidine pulse technique and Hoechst-Giemsa differential staining of metaphase chromosomes. This comparison indirectly shows the S-phase of the heteroploid cells of V-79 to be 2 h shorter than the diploid cell S-phase. When the thymidine pulse is applied to diploid lung fibroblasts at mid-S-phase, differential staining colours metaphase chromosomes a pale blue. Performing the corresponding experiment with V-79 cells, neither a pale blue nor dark red staining is obtained, but rather an intermediate shade, showing prominently dark staining regions in parts. The pause in DNA synthesis observed at mid-S-phase of the diploid Chinese hamster lung fibroblasts seems to be omitted at mid-S-phase of the V-79 cells.     

EFFECT OF METHOTREXATE ON THE CELL CYCLE OF L1210 LEUKEMIA

The influence of methotrexate (MTX) on the proliferative activity of cells in different phases of cell cycle has been studied. MTX (5 mg/kg) was injected i.p. 3 days after the inoculation of 5 × 10⁶ leukemia cells into F₁ (DBA × C57 BL) mice. It was shown that MTX causes degeneration of cells, being in G₁- as well as in S-phase at the time of drug injection. Incorporation of ³H-TdR was suppressed for a period ranging from 2 to 12 hr after MTX administration, which is demonstrated by the decrease in the number of grains per cell. The number of cells labeled after ³H-TdR injection was also sharply decreased during this period. For a period of 3 until 15 hr after MTX administration the mitotic index decreased significantly as a result of inhibition of DNA synthesis. The blocking of the G₁-S transition was evident during 4 hr after MTX. Thereafter the G₁-S transition proceeds at a rate which is practically equal to that for nontreated controls. MTX did not inhibit transition to mitosis of cells being in G₂-phase and in a very late S-phase at the time of drug injection. The sensitivity of G₁-cells to the cytocidal effect of MTX shows that for L1210 leukemia cells MTX can be classified as a cycle-specific drug killing both G₁ and S-cells rather than S-phase specific agent with self-limitation.     

Significance of treatment interval and DNA repair in the enhancement of viral transformation by chemical carcinogens and mutagens

Treatment of Syrian hamster embryo cells with diverse classes of chemical carcinogens enhanced transformation by a carcinogenic simian adenovirus, SA7. Optimal enhancement was a function of time of chemical addition in relation to time of virus addition and cell transfer. Aflatoxin B₁ (AFB₁) and the polycyclic hydrocarbons, benzo(a)pyrene (B(a)P), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz(a)anthracene (DMBA) enhanced SA7 transformation when added prior to virus, but inhibited transformation when added after virus adsorption and cell transfer. The enhancement of SA7 transformation was maximal when cytosine arabinoside, caffeine and 6-acetoxy-benzo(a)pyrene (6-ac-B(a)P) were added after virus, but minimal when added before virus. A third class of chemicals, including β-propiolactone (β-PL), methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (Ac-AAF), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and methylazoxymethanol acetate (MAM-ac), enhanced SA7 transformation added before, or after, virus inoculation and cell transfer. All chemicals, which induced changes in DNA sedimentation in alkaline sucrose gradients and unscheduled DNA (repair) synthesis in hamster cells, increased the frequency of SA7 transformation. However, several chemicals such as dibenz(a,h)anthracene (DB(a,h)A), benzo(e)pyrene (B(e)P), cytosine arabinoside, and caffeine enhanced SA7 transformation but did not induce DNA sedimentation changes or repair. Chemicals that cause DNA damage, which can be repaired by hamster cells, may enhance viral transformation by providing additional sites for integration of viral DNA during the repair process. Chemicals that apparently do not induce DNA repair synthesis may enhance viral transformation by incorporation of viral DNA into gaps in cell DNA at sites of unrepaired damage during scheduled DNA synthesis.     

Inhibition of H-thymidine incorporation by hydroxyurea: Atypical response of mycoplasma-infected cells in culture

Contamination with Mycoplasma hyorhinis was demonstrated in long-term cultures of HeLa, BICR/M1R_K rat mammary tumor, and NV1C rat neurinoma cells, by microbiological, equilibrium sedimentation, and autoradiographic techniques. In non-infected DNA-synthesizing cells, hydroxyurea (HU) in concentrations 10⁻⁴ M typically inhibits ³H-thymidine (³H-TdR) incorporation into acid-insoluble material. This effect was lacking in the contaminated cell lines, although HU did block nuclear DNA replication, as shown by pulse-cytophotometric analyses. The response to HU could be restored to normal by supplementing the culture medium either with the anti-mycoplasma agent Tylosin or with fresh rat serum. The total ³H-activity in non-infected (or anti-mycoplasma treated) versus infected cells, in the absence of HU, was up to four times higher in the former. The data indicate that (i) incorporation of ³H-TdR into the nuclear DNA of contaminated cells was strongly reduced, probably due to a ‘scavenger effect’ (i.e. utilisation and rapid cleavage) by the mycoplasma; (ii) mycoplasmal ³H-TdR incorporation, contrary to nuclear DNA replication, was insensitive to HU in concentrations 10⁻² M. If equally valid for other species of mycoplasma, the observed phenomenon provides a criterion (together with the possibility of a rapid test) for the presence of mycoplasmal contamination in cell cultures.     

Inhibition of 3H-thymidine incorporation by hydroxyurea: atpical response of mycoplasma-infected cells in culture.

Contamination with Mycoplasma hyorhinis was demonstrated in long-term cultures of HeLa, BICR/M1R_K rat mammary tumor, and NV1C rat neurinoma cells, by microbiological, equilibrium sedimentation, and autoradiographic techniques. In non-infected DNA-synthesizing cells, hydroxyurea (HU) in concentrations ? 10^?4 M typically inhibits ³H-thymidine (³H-TdR) incorporation into acid-insoluble material. This effect was lacking in the contaminated cell lines, although HU did block nuclear DNA replication, as shown by pulse-cytophotometric analyses. The response to HU could be restored to normal by supplementing the culture medium either with the anti-mycoplasma agent Tylosin or with fresh rat serum. The total ³H-activity in non-infected (or anti-mycoplasma treated) versus infected cells, in the absence of HU, was up to four times higher in the former. The data indicate that (i) incorporation of ³H-TdR into the nuclear DNA of contaminated cells was strongly reduced, probably due to a ‘scavenger effect’ (i.e. utilisation and rapid cleavage) by the mycoplasma; (ii) mycoplasmal ³H-TdR incorporation, contrary to nuclear DNA replication, was insensitive to HU in concentrations ? 10^?2 M. If equally valid for other species of mycoplasma, the observed phenomenon provides a criterion (together with the possibility of a rapid test) for the presence of mycoplasmal contamination in cell cultures.     

THE EFFECTS OF CYTOSINE ARABINOSIDE ON THE CELL CYCLE OF CULTURED HUMAN LEUCOCYTES: A MICRODENSITOMETRIC AND AUTORADIOGRAPHIC STUDY

PHA-stimulated leucocytes treated with cytosine arabinoside showed a changed uptake of [³H]thymidine, a lowered mitotic index and chromosome damage. Combined autoradiography and Feulgen microdensitometry demonstrated inhibition of DNA synthesis. Cells in the S period of the cell cycle were arrested in their progress, and cells newly entering S accumulated at the beginning of this period. At stronger concentrations these effects on the cell cycle were complicated by the cytotoxic effect of cytosine arabinoside. The inhibition of DNA synthesis is considered in the light of the chromosomal damage and megaloblastic changes caused by cytosine arabinoside, and is compared to the different pattern of DNA arrest found in megaloblastic anaemia.     

Inhibition of 3H-thymidine incorporation of lymphocytes by a soluble factor from macrophages

Supernatants of peritoneal macrophages cocultivated with spleen cells or thymocytes contain a factor suppressing the ³H-thymidine (³H-TdR) incorporation of PHA-stimulated lymphocytes. The suppressing activity is not due to cytotoxicity and does not affect the rate of cell transformation or cell proliferation. The factor reduces only the ³H-TdR incorporation and not the DNA synthesis of lymphocytes. It does not show target cell specificity. The factor is dialysable, heat stable, and generated by macrophages.     

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoeleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1⁰ protein was predominant in G₀ cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.     

Effect of cytosine arabinoside on replicon initiation in human lymphoblasts

Cytosine arabinoside inhibited DNA synthesis in human lymphoblasts by inhibiting the initiation of DNA replication units. This effect was observed by a decrease in the incorporation of (³H) thymidine into low molecular weight DNA analyzed by velocity sedimentation in alkaline sucrose gradients. In contrast, there was no detectable effect on chain elongation and joining of those molecules that initiated replication before addition of the drug. These data indicate that cytosine arabinoside acts preferentially at the level of initiation of DNA replication rather than chain elongation.     

Isolation and cell cycle analysis of temperature-sensitive mutants from chinese hamster cells

HeLa cell mitochondria were allowed to incorporate ³H-thymidine in a cell free system and the effect of ethidium bromide, cytosine arabinoside and cytosine arabinoside triphosphate on the labeling of mitochondrial DNA was studied. The labeled products, isolated by sedimentation velocity in CsCl-ethidium bromide two-step gradients, showed similar sedimentation profiles as in vivo labeled mtDNA. Cytosine arabinoside triphosphate and ethidium bromide strongly inhibited the labeling of mitochondrial DNA, whereas cytosine arabinoside appeared to be much less effective. Tritiated deoxycytidine was found to be incorporated by isolated mitochondria, whereas cytosine arabinoside was shown to enter the mitochondrial acid-soluble pool but not to be incorporated in acid-insoluble form. These results are in agreement with the previously reported findings of in vivo experiments.     

AUTOSYNCHRONIZATION OF RAT LIVER CELLS WITH ENDOGENOUS CORTICOSTERONE AFTER PARTIAL HEPATECTOMY

After adrenalectomy in adult male rats ³H-TdR incorporation into the liver parenchymal cells is increased 4–8 times and the mitotic index rises from 0–31 % to 1–3%; this is inhibited by corticosterone. After hepatectomy the serum corticosterone level increases from 18 μg/100 ml to 57 μg/100 ml. The corticosterone binding capacity of the serum declines from 2–06 to 0–17. The activity of tyrosine transaminase doubles, whereas the incorporation of ³H-TdR into the liver cells is decreased by a factor of 5–7. Thereafter the binding capacity increases again and reaches, 24 hr after operation, a value of 3–82. The tyrosine transaminase activity and the serum corticosterone content return to normal. ³H-TdR incorporation, however, increases by a factor of 7-7 of the initial value. We concluded that in the first few hours after partial hepatectomy corticosterone blocks the liver cells in G₁ and an accumulation of the cells occurs at this cell cycle phase. Folio wing the binding of the corticosterone by serum proteins a little later the liver cells enter the S-phase synchronously.     

Cell Cycle activation and ouabain-sensitive ion movements of 3T3 and C3H-10T½ fibroblasts

The introduction of either PGF_2α (10^?7 M) or TPA (10^?7 M) stimulated, ouabain-sensitive ⁸⁶Rb⁺ influx at 30 min in postconfluent 3T3-4 mouse fibroblast cultures by 117% and 124%, respectively. Both TPA and PGF_2α at these concentrations stimulated the incorporation of ³H-TdR into DNA. TPA had the greatest stimulatory effect, which was similar to that obtained with 10% fetal calf serum. In accord with the idea that modulation of membrane processes such as Na⁺/K⁺ pump activity in fibroblasts may reflect important events related to the initiation of DNA synthesis, it was observed that in both 3T3-4 and C3H-1 0T½ cells there were parallel increases in ³H-TdR incorporation and ouabain-sensitive ⁸⁶Rb⁺ influxes with 10^?7 M TPA, whereas PGF_2α stimulated a significant increase in ³H-TdR incorporation in 3T3-4 but not C3H-10T½ cells and only marginal increases in ouabain-sensitive ⁸⁶Rb⁺ influx in both. Therefore, although there appears to be a close correlation between Na⁺/K⁺ pump activation and subsequent S-phase entry following TPA stimulation, a similar correlation for PGF_2α cannot be confirmed.     

DIFFERENT ACTION OF SUICIDAL DOSES OF TRITIATED THYMIDINE AND HYDROXYUREA ON MURINE HAEMOPOIETIC CELLS

In order to gather information on the factors that cause the different action of suicidal doses of tritiated thymidine (³H-TdR) and of hydroxyurea on murine stem cells, the incorporation of ³H-TdR into DNA of bone marrow and spleen cells has been studied. Continuous death of labelled cells after suicidal ³H-TdR is indicated by a more pronounced decline of total DNA-bound radioactivity in bone marrow and spleen cells compared to that in control animals which had received tracer doses of ³H-TdR. Extensive and rapid loss of DNA-bound radioactivity occurred in ³H-TdR labelled animals after hydroxyurea treatment indicating an instantaneous and highly effective killing of labelled cells. After double labelling of DNA with ³H-TdR and ¹²⁵iodo-deoxyuridine (¹²⁵I-UdR), the decline of the ratio of DNA-bound ¹²⁵I to DNA-bound ³H after suicidal ³H-TdR indicates prolonged tritium reutilization. Following hydroxyurea, reutilization was completed within the first 12 hr after drug administration. These findings explain in part the slow recovery of different stem cell compartments after suicidal ³H-TdR on the basis of protracted tritium reutilization as compared to the fast recovery which follows the rapid action of hydroxyurea.     

Inhibition of DNA Synthesis but not of Poly-dAT Synthesis by the Arabinose Analogue of Cytidine <Emphasis Type="Italic">in vitro</Emphasis>

Kimball and Wilson¹ reported that the arabinose analogue of cytidine (ara-C) inhibited DNA polymerase in a crude extract prepared from Ehrlich ascites cells. Furth and Cohen² observed cytosine arabinoside triphosphate (ara-CTP) inhibited DNA polymerase in extracts from either calf thymus or bovine lymphosarcoma tissue, although these investigators³ had already found no effect of ara-CTP on DNA polymerase from Escherichia coli. The inhibition in both of these cases could be substantially reversed by dCTP; but incorporation of the arabinose nucleotide (ara-CMP) into DNA could not be unequivocally demonstrated. Graham and Whitmore⁴ reported the incorporation of ara-C into DNA in vivo and the inhibition of a DNA polymerase from L cells by ara-CTP. They found that ara-CMP was initially incorporated into small DNA strands but subsequently appeared in long strands. Momparler⁵ has presented evidence that, in vitro, ara-C incorporation was limited to the 3′-hydroxyl end of DNA chains. Such incorporation might be expected to block further chain elongation but this expectation was not supported by the evidence presented by Graham and Whitmore.     

Detection of cytosine arabinoside resistant cells at low frequency using the bromodeoxyuridine/DNA assay

This report describes a rapid and sensitive procedure for detection of cytosine arabinoside- (Ara-C) resistant mouse leukemia cells (L1210) in a predominantly Ara-C-sensitive population. L1210 cell lines sensitive or resistant to Ara-C were grown and treated with Ara-C in vitro or in vivo. Ara-C-resistant cells were detected as those cells with S-phase DNA content retaining the ability to incorporate bromodeoxyuridine (BrdUrd) after treatment with Ara-C. The BrdUrd incorporation ability of the S-phase cells was assessed by simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated BrdUrd. The proportion of Ara-C-resistant cells was accurately estimated at frequencies approaching 10(-3).     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

Effect of thiopyrimidine ribonucleosides on DNA and RNA synthesis in synchronized mammalian cell cultures

The uptake of ³H-uridine into RNA and of ³H-thymidine into DNA was investigated in synchronized Chinese hamster cells which had been exposed to thiopyrimidine ribonucleosides. The cells were synchronized at metaphase by reversal of colcemid inhibition; these cells were then labeled with either ³H-thymidine or ³H-uridine at selected times, and analyzed in autoradiographs. Incorporation of ³H-thymidine into DNA was not inhibited by administration to the cells of 2-thiouridine or 4-thiouridine (4 × 10⁻³ M). Exposure of the cells to the anti-metabolites for over 15 h significantly reduced the incorporation of ³H-uridine into nuclear RNA and completely blocked the labeling of cytoplasmic RNA. This finding is interpreted as an indication that RNA synthesis was inhibited in cells which continued to synthesize DNA. The inhibition of RNA synthesis hindered cell division and decreased cell viability. This lethal effect is similar to the “unbalanced growth” induced by inhibitors of DNA synthesis. The thiopyrimidine ribonucleosides, however, killed mammalian cells without inhibiting DNA synthesis.