Prolonged Cell Cycle Research Articles

Cell cycle duration in antheridial filaments ofChara spp. (Characeae) with different genome size and heterochromatin content

The relationship between nuclear 1 C DNA content and cell cycle progression throughout successive stages of antheridial filaments were studied among five taxa ofChara: two dioecious species (n = 14):C. aspera (7.2 pg DNA),C. tomentosa (7.4 pg DNA), and three monoecious species (n = 28):C. vulgaris (13.5 pg DNA),C. fragilis (19.3 pg DNA), andC. contraria (19.6 pg DNA). With the use of double3H-thymidine labelling and morphometry a number of characteristics common to all of the investigated species were determined within the “proliferative periods” preceding spermiogenesis. These include: (1) simplified type of the cell cycle (S + G2 + M), due to complete lack of G1 intervals, (2) constant duration of S phase, (3) progressive shortening of G2 + M periods, and (4) gradual reduction of cell lengths at successive mitotic divisions. Nucleotypic dependence was found between genome size and several time parameters estimated for consecutive stages of antheridial filaments: the higher the DNA C-value, the longer the cell cycles, their component phases, the total duration of “the proliferative period”, as well as the lower the rate of growth of interphase cells. Differential Giemsa staining of late G2 phase nuclei revealed that the higher content of C-heterochromatin is connected with prolonged cell cycle durations in species with similar DNA C-values.

Molecular cloning and functional analysis of three subunits of yeast proteasome.

The genes encoding three subunits of Saccharomyces cerevisiae proteasome were cloned and sequenced. The deduced amino acid sequences were homologous not only to each other (30 to 40% identity) but also to those of rat and Drosophila proteasomes (25 to 65% identity). However, none of these sequences showed any similarity to any other known sequences, including various proteases, suggesting that these proteasome subunits may constitute a unique gene family. Gene disruption analyses revealed that two of the three subunits (subunits Y7 and Y8) are essential for growth, indicating that the proteasome and its individual subunits play an indispensable role in fundamental biological processes. On the other hand, subunit Y13 is not essential; haploid cells with a disrupted Y13 gene can proliferate, although the doubling time is longer than that of cells with nondisrupted genes. In addition, biochemical analysis revealed that proteasome prepared from the Y13 disrupted cells contains tryptic and chymotryptic activities equivalent to those of nondisrupted cells, indicating that the Y13 subunit is not essential for tryptic or chymotryptic activity. However, the chymotryptic activity of the Y13 disrupted cells is not dependent on sodium dodecyl sulfate (SDS), an activator of proteasome, since nearly full activity was observed in the absence of SDS. Thus, the activity in proteasome of the Y13 disrupted cells might result in unregulated intracellular proteolysis, thus leading to the prolonged cell cycle. These results indicate that cloned proteasome subunits having similar sequences to the yeast Y13 subunit are structural, but not catalytic, components of proteasome. It is also suggested that two subunits (Y7 and Y8) might occupy positions essential to proteasome structure or activity, whereas subunit Y13 is in a nonessential but important position.

Tumor cytokinetic effects of acute starvation versus polyamine depletion in tumor‐bearing mice

Previous investigations in our laboratory have demonstrated that both acute host starvation and polyamine depletion by means of the irreversible ODC-inhibitor (ODC = ornithine-decarboxylase) fluoro-methylornithine (DFMO) lead to pronounced growth retardation of rapidly proliferating tumors. The aim of this investigation was to elucidate how these different interventions affect cell kinetics and cell cycle phases in vivo. Adult nongrowing mice (C57Bl/J) bearing a poorly differentiated rapidly growing methylcholanthrene induced sarcoma were used. Combined measurements of bromodeoxyuridine incorporation into DNA and flow cytometric techniques were used. Starvation and DFMO treatment resulted in a prolonged cell cycle transit compared to freely fed animals. Tumor cells from DFMO-treated mice demonstrated an increased time for DNA synthesis and a relatively larger accumulation of cells in the G2M phase, whereas tumor cells from starved animals were accumulated in the G0G1 phase. The fractional cell loss of tumor cell during proliferation was calculated to be around 18% higher in DFMO-treated animals compared to starved and freely fed tumor-bearing mice. This study demonstrates that different mechanisms are involved in tumor growth suppression from substrate deficiency (starvation) and from inhibition of polyamine synthesis.

Molecular Cloning and Functional Analysis of Three Subunits of Yeast Proteasome

The genes encoding three subunits of Saccharomyces cerevisiae proteasome were cloned and sequenced. The deduced amino acid sequences were homologous not only to each other (30 to 40% identity) but also to those of rat and Drosophila proteasomes (25 to 65% identity). However, none of these sequences showed any similarity to any other known sequences, including various proteases, suggesting that these proteasome subunits may constitute a unique gene family. Gene disruption analyses revealed that two of the three subunits (subunits Y7 and Y8) are essential for growth, indicating that the proteasome and its individual subunits play an indispensable role in fundamental biological processes. On the other hand, subunit Y13 is not essential; haploid cells with a disrupted Y13 gene can proliferate, although the doubling time is longer than that of cells with nondisrupted genes. In addition, biochemical analysis revealed that proteasome prepared from the Y13 disrupted cells contains tryptic and chymotryptic activities equivalent to those of nondisrupted cells, indicating that the Y13 subunit is not essential for tryptic or chymotryptic activity. However, the chymotryptic activity of the Y13 disrupted cells is not dependent on sodium dodecyl sulfate (SDS), an activator of proteasome, since nearly full activity was observed in the absence of SDS. Thus, the activity in proteasome of the Y13 disrupted cells might result in unregulated intracellular proteolysis, thus leading to the prolonged cell cycle. These results indicate that cloned proteasome subunits having similar sequences to the yeast Y13 subunit are structural, but not catalytic, components of proteasome. It is also suggested that two subunits (Y7 and Y8) might occupy positions essential to proteasome structure or activity, whereas subunit Y13 is in a nonessential but important position.

Cytostatic effects of radiographic contrast media in synchronized cell cultures.

The cytostatic effects of conventional high osmolal ionic contrast media (meglumine-calcium metrizoate and Na-metrizoate) and new low osmolal nonionic contrast media (iohexol and iopamidol) in synchronized cell cultures were tested. The cell-cycle prolongation was most pronounced when the contrast media were added in the G1 phase, but there was also a marked effect when the contrast media were added in the S phase or late in the G2 phase. The cytostatic effect even persisted into the first cell cycle following the termination of the exposure. All four contrast media exerted effects stronger than that of equiosmolal saline. Iohexol and iopamidol produced a more severe effect than meglumine-calcium metrizoate and Nametrizoate at equal osmolality. Thus, the cytostatic effect of contrast media cannot be explained only by hypertonicity; the contrast media must have an additional specific cytostatic effect. When the cytostatic effect was related to iodine concentration, the new low osmolal nonionic contrast media influenced the cell cycle less than the conventional high osmolal ionic contrast media.

Retardation of cell cycle progression in yeast cells recovering from DNA damage: a study at the single cell level.

Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.

Cell cycle traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage.

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.

The delta agent comes of age.

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent anti-mitotic components of natural origin but it is however intrinsically unstable. A novel series of CA-4 analogues incorporating a 3,4-diaryl-2-azetidinone (β-lactam) ring were designed and synthesised with the objective to prevent cis-trans isomerisation and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected β-lactam CA-4 analogues demonstrated potent anti-tubulin, anti-proliferative and anti-mitotic effects in human leukemia cells. A lead β-lactam analogue, CA-476 displayed comparable antiproliferative activities with CA-4. CA-476 induced rapid apoptosis in HL-60 acute myeloid leukemia (AML) cells which was accompanied by depolymerisation of the microtubular network, PARP cleavage, caspase-3 activation and Bcl-2 cleavage. A prolonged G2M cell cycle arrest accompanied with a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the anti-apoptotic proteins Bcl-2 and Bcl-xL preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and β-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-476 induced significantly more apoptosis compared to imatinib mesylate in ex-vivo CML patient samples including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogues of CA-4 have been designed and synthesised which display significant clinical potential as anti-leukemic agents.

Increased sister chromatid exchanges in human cell lines characterized by monosomy X or structural abnormalities of the X chromosome.

In the present investigation we test the hypothesis that deficiencies of the X chromosome affect sister chromatid exchange (SCE) frequencies in human fibroblast cell lines. Our results show increased mean SCE frequencies in cell lines with abnormalities of the X chromosome: 45,X; 46,X,del(X) (q13), 46,X,del(X)(p11), and 46,X,i(Xq); control cell lines were 46,XX. In only one abnormal line [46,X,del(X)(p11)] was the increase not significant after correcting for multiple comparisons. If SCE formation is replication-dependent, the increased SCE frequencies might merely reflect the prolonged cell cycle we reported previously in cell lines with X chromosome abnormalities (Simpson and LeBeau 1981). Other explanations for differences between cell lines are possible, e.g., that deleted loci on the X chromosome affect cellular uptake of bromodeoxyuridine (BrDU). However, specific mechanisms were not explored directly.

Effects of Ethanol on Cell Volume and Protein Synthesis in a Human Lymphoblastoid Cell Line (Raji)

A human lymphoblastoid cell line (Raji) developed macrocytosis after 5-7 days when cultured in the presence of 100, 250 and 500 mg ethanol/dl. The degree of macrocytosis was least with 100 mg/dl and greatest with 500 mg/dl. The macrocytosis was associated with a proportionate increase in the total protein content per cell, was reversed after culture in the absence of ethanol and was uninfluenced by the supplementation of the culture medium with 50 micrograms folic or folinic acids per millilitre. Ethanol also caused a substantial prolongation of the cell doubling time at concentrations of 250 and 500 mg/dl (but not 100 mg/dl) and this was associated with some increase in the proportion of non-viable cells in the cultures. Furthermore, ethanol increased the incorporation of 3H-leucine into protein per femtolitre of cell volume. It is proposed that the ethanol-induced macrocytosis may have developed as a consequence of the stimulation of the rate of protein synthesis within a normal or prolonged cell cycle.

The role of protein metabolism in glucocorticoid-induced prolongation of G1 phase in human NHIK 3025 cells.

It has previously been found that human NHIK 3025 cells have a glucocorticoid-sensitive restriction point in mid-G1 phase of the cell cycle. When these cells were synchronized by mitotic selection and exposed to dexamethasone before the restriction point, G1 phase was prolonged whereas the rest of the cell cycle was unperturbed by the hormone. These observations were confirmed by flowcytometric measurements of synchronized cells in the present study. Cells that received dexamethasone (10(-6) M) just after mitotic selection had a 4 hour prolongation of both G1 and the total cell cycle. However, the general rates of both protein synthesis and protein degradation were found not to be altered by the hormone, i.e., the rate of protein accumulation in dexamethasone exposed cells was equal to that of control cells. Dexamethasone exposed NHIK 3025 cells were found to be larger than control cells at the time of cell division. This is a direct consequence of a prolonged cell cycle duration with no change in general protein metabolism. It thus appears that the dexamethasone-induced prolongation of G1 phase is the result of a steroid-regulated G1 specific process(es) leading toward DNA replication, a process that does not alter general protein accumulation.

On the existence of non‐cycling germinative cells in human epidermis in vivo and cell cycle aspects of psoriasis

Abstract. This report deals with the controversies of whether all germinative epidermal cells in human epidermis are in the cycling state and whether stimulated hyperproliferation of psoriatic epidermis is due to a shortening of the cell cycle time or to a recruitment of non‐cycling germinative epidermal cells. Experiments were performed on human subjects in vivo. Continuous infusion of [3H]thymidine for 8½ days indicated that 40% of germinative epidermal cells reside in the non‐cycling state. Proliferative stimulation by tape stripping indicated recruitment of non‐cycling (G0) germinative epidermal cells in both normal and psoriatic skin, and a prolongation (rather than a shortening) of cell cycle traverse in activated psoriatic epidermal cells.

Sister chromatid differentiation and cell‐cycle‐specific patterns in chronic myelocytic leukemia and normal bone marrow

After in vitro incubation with bromodeoxyuridine (BrdUrd)-containing medium, metaphases of Philadelphia (Ph1)-chromosome positive cells in the blood and/or bone marrow of eight untreated patients with chronic myelocytic leukemia (CML) were analysed by means of sister chromatid differentiation (SCD) and compared to 10 normal bone marrows. The majority of proliferating Ph1-positive cells (range: 46-86%) were unable to complete more than one cell cycle during the culture period of 50 h (MI metaphases). Ph1-positive M2 metaphases which had completed two cell cycles were found in the range of 18-54% and almost no M3 metaphases were detected after passage through three cell cycles (0-1%). The corresponding findings in proliferating normal bone-marrow cells were: MI (13-68%), M2 (30-79%) and M3 (0-18%). These data support the notion that there is a prolonged cell cycle time in CML which can be measured by SCD, demonstrating a proportional shift from M3/M2 to MI metaphases. SCD is suggested for kinetic studies on human malignant cell clones characterized by marker chromosomes.

Effect of temperature variation on sister-chromatid exchange and cell-cycle duration in cultured human lymphocytes

The relation of temperature variation and cell-cycle duration and their influence on SCE frequencies in human peripheral blood lymphocytes was studied. The results indicate that the frequency of SCEs is affected by variation in temperature. Either increase or decrease in the incubation temperature of cells leads to a higher frequency of SCEs than in control cultures grown at 37°C. With a decrease in temperature to 35°C the increase in SCE is accompanied by prolonged cell cycle. No significant difference in cell cycle was observed with the increase of temperature to 39°C.

Cell population kinetics of thyroid follicular cells in infant rats.

T cell population kinetics of thyroid follicular cells in rats were studied by means of autoradiography and a statmokinetic technique. During the first fortnight after birth no significant changes in the mitotic index (MI) and labelling index (LI) were found. In the next 2 weeks a constant decrease in the number of proliferating cells occurs. In 10-day old animals 40% of the follicular cells were in the cell cycle (GF); 3.25 +/- 0.77 (SEM) % in the S phase and 0.18 +/- 0.04% in mitoses (MI). Day-night changes in the LI and mitotic rate (MR) indicated a peak value at 13.30 hours with a lowest value at 22.30 hours. The mean LI and MR averaged over the whole 24 hr were 3.1 +/- 0.1% and 122.2 +/- 18.1%, respectively. In 10-day old animals, using the fraction of labelled mitoses (FLM) method the median cell cycle time (TC) was 79 hr and the phase durations were TG1--64.6 hr, TS--8.2 hr and TG2--5.1 hr. The decrease in the number of proliferating cells with the age of the animals is considered to be a result of both cell cycle prolongation and in growth fraction reduction.

Gonadal and statural determinants on the X chromosome and their relationship to in vitro studies showing prolonged cell cycles in 45,X; 46,X,del(X)(p11); 46,X,del(X)(q13); and 46,X,del(X)(q22) fibroblasts

Correlation of clinical features with cytogenetic abnormalities for individuals showing deletions of the X short arm (Xp) or the X long arm (Xq) indicate the following: (1) both Xp and Xq are necessary to assure normal ovarian development, although (2) persisting ovarian function is not infrequently associated with either (del(X)(p11) or del(Xq)(13,21,22, or 24). (3) Ovarian determinants on Xp are localized to region Xp11, but determinants on Xq cannot be precisely localized. (4) Both Xp and Xq contain statural determinants, the former localized to region Xp21 leads to Xpter. Both cell generation time and phases of the cell cycle were studied to test the hypothesis that the short stature, intrauterine growth retardation, and high embryonic lethality of 45,X can be explained on the basis of intrinsic retardation of cell division (i.e., prolonged cell cycle). Cell generation times of four 45,X fibroblast lines were significantly longer than those of for normal diploid lines, a difference accounted for by a prolonged S phase. 46,X,del(X)(p11), 46,X,del(X)(q13), and 46,X,del(X)(q22) lines also showed increased cell generation times when compared to 46,XX lines.

The role of protein accumulation in the cell cycle control of human NHIK 3025 cells.

The cell cycle kinetics of NHIK 3025 cells, synchronized by mitotic selection, was studied in the presence of cycloheximide at concentrations (0.125-1.25 microM) which inhibited protein synthesis partially and slowed down the rate of cell cycle traverse. The median cell cycle duration was equal to the protein doubling time in both the control cells and in the cycloheximide-treated cultures at all drug concentrations. This conclusion was valid whether protein synthesis was continuously depressed by cycloheximide throughout the entire cell cycle, or temporarily inhibited during shorter periods at various stages of the cell cycle. These results may indicate that cell division does not take place before the cell has reached a critical size, or has completed a protein accumulation-dependent sequence of events. When present throughout the cell cycle, cycloheximide increased the median G1 duration proportionally to the total cell cycle prolongation. However, the entry of cells into S, once initiated, proceeded at an almost unaffected rate even at cycloheximide concentrations which reduced the rate of protein synthesis 50%. The onset of DNA synthesis seemed to take place in the cycloheximide-treated cells at a time when the protein content was lower than in the control cells. This might suggest that DNA synthesis in NHIK 3025 cells is not initiated at a critical cell mass.

Study of selected characteristics of theChlamydomonas reinhardii strains with altered sensitivity to UV radiation

Selected characteristics and streptomycin resistance were studied in a UV radiation sensitive (UVS1) and a UV radiation resistant (UVR1) strains, and the data were compared with results obtained with an original type strain. A partial prolongation of the cell cycle in the UVR1 strain as compared with the original type strain could be observed in studying cell volume growth, cell numbers, DNA, RNA and protein synthesis during the synchronous cycle. Under these conditions, the UVS1 strain behaved as a temperature sensitive cell cycle mutant. In inducing streptomycin resistant mutants, the highest frequencies in various doses were recorded in the UVS1 strain.

Effects of steroids and different culture media on cell cycle of the androgen-sensitive human cell line NHIK3025.

The median cell cycle of synchronized NHIK3025 cells grown in Eagle's MEM with 10% foetal calf serum was 23.6 h, compared to 18 h observed earlier in Puck's E2a medium with 30% serum (20% human and 10% horse). This difference is due to prolongation of both G1 and G2 in the MEM type medium. Testosterone and 4-androstene-3 beta, 17 beta-diol reduced the cell cycle of synchronized populations of NHIK3025 cultured in MEM type medium. Dexamethasone gave cell cycle prolongation while estradiol had no effect. These results are in accordance with steroid-induced changes in growth of asynchronous cell populations observed earlier. The androgen growth stimulation was partly due to a shortening of G2 but was not exclusively located in one particular phase of the cell cycle.

Cell-cycle inhibition by misonidazole of human cells cultivated in vitro under aerobic conditions.

By means of flow cytometric recording of DNA histograms and counting of cells in synchronized populations, we have found that misonidazole (MIS) in clinically relevant concentrations induces cell-kinetic changes in human cells (NHIK 3025) cultivated in vitro under aerobic conditions. The effect seems to be a general lengthening of the cell cycle, affecting all phases. However, induction of this effect is phase-dependent, since only cells exposed to MIS during mitosis and/or early G1 will suffer significant cell-cycle prolongation. In exponentially growing populations this effect of MIS leads to a transient increase in the fraction of G1 cells and a corresponding decrease in the fraction of S cells. The possible significance of this effect for the clinical use of MIS is discussed.