The "protective" effect of gamma-irradiation of cells in metaphase of mitosis following V-irradiation during the S period]

20,1% cells with chromosomes aberrations were obtained after UV-irradiation of embryonal fibroblasts of mice at the S-stage in vitro at a decreasing dose of 40erg/mm2. Subsequent gamma-irradiation at the metaphase of the first mitosis at a 5 krad dose led to a statistically significant decrease of the frequency of aberrant cells observed in the same mitosis down to 11,7%. The frequency of spontaneous aberrations did not change during the first few minutes after gamma-irradiation of intact cells at the metaphase. The "protective" effect of gamma-rays can not be explained either by unequal changes of the duration of mitotic stages for aberrant and normal cells, or by sticking of chromosome fragments or by breaks of bridges at the anaphase. The death of cells "under irradiation" also appears to be a hardly probable case of the effect observed. It is assumed that the decrease of the aberrations frequency is the result of predicted earlier modification of the processes of realization of potential chromosome damages into visible aberrations at the metaphase.     

The synthetic purine reversine selectively induces cell death of cancer cells

The synthetic purine reversine has been shown to possess a dual activity as it promotes the de‐differentiation of adult cells, including fibroblasts, into stem‐cell‐like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti‐cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced. J. Cell. Biochem. 113: 3207–3217, 2012. © 2012 Wiley Periodicals, Inc.     

Effect of 2-mercaptoethanol on the individual periods of the mitotic cycle

Dynamics of the mitotic cycle of the KEPV cells being on different interphase stages at the start of a 20 hour 2-mercaptoethanol (0.001 M) treatment has been studied during the treatment and for 11 hours after washing out the agent. The KEPV cells affected by mercaptoethanol during the interphase (G1, S, G2) were shown to continue their passage through the cycle to enter mitosis, but part of the cells of the S period and of the first half of the G2 period were arrested in the interphase. In the presence of mercaptoethanol, mitotic cells reach the metaphase stage, and their further behaviour depends on the duration of the treatment. For the first 8 hours of treatment, a phase of "unstable block" exists for cells that were in S and G2 periods at the beginning of treatment, while other cells are transformed into K-metaphases. 8 hours later a phase of "stable block" occurs and all the normal metaphases are transformed into K-metaphases. After washing out the culture from mercaptoethanol the cells are ejected from the block in K-metaphase. The transformation from K-metaphase into the normal metaphase is realised in the course of this process. The cells which were in S and G2 periods at the beginning of the treatment are ejected from the block simultaneously after washing, while the cells of the G1 period--with a small delay. After washing out mercaptoethanol the cells that were in the interphase (G1, S, G2) at the beginning of the treatment are capable of producing both multipolar mitoses and mitoses without cytotomy.     

Tumor Treating Fields Perturb the Localization of Septins and Cause Aberrant Mitotic Exit

The anti-tumor effects of chemotherapy and radiation are thought to be mediated by triggering G₁/S or G₂/M cell cycle checkpoints, while spindle poisons, such as paclitaxel, block metaphase exit by initiating the spindle assembly checkpoint. In contrast, we have found that 150 kilohertz (kHz) alternating electric fields, also known as Tumor Treating Fields (TTFields), perturbed cells at the transition from metaphase to anaphase. Cells exposed to the TTFields during mitosis showed normal progression to this point, but exhibited uncontrolled membrane blebbing that coincided with metaphase exit. The ability of such alternating electric fields to affect cellular physiology is likely to be dependent on their interactions with proteins possessing high dipole moments. The mitotic Septin complex consisting of Septin 2, 6 and 7, possesses a high calculated dipole moment of 2711 Debyes (D) and plays a central role in positioning the cytokinetic cleavage furrow, and governing its contraction during ingression. We showed that during anaphase, TTFields inhibited Septin localization to the anaphase spindle midline and cytokinetic furrow, as well as its association with microtubules during cell attachment and spreading on fibronectin. After aberrant metaphase exit as a consequence of TTFields exposure, cells exhibited aberrant nuclear architecture and signs of cellular stress including an overall decrease in cellular proliferation, followed by apoptosis that was strongly influenced by the p53 mutational status. Thus, TTFields are able to diminish cell proliferation by specifically perturbing key proteins involved in cell division, leading to mitotic catastrophe and subsequent cell death.     

Comparative effects of ftorafur and 5-fluorouracil on DNA synthesis in rat small intestine.

The effect of equimolar doses of ftorafur (100 mg/kg) and 5-fluorouracil (65 mg/kg) on the $\text{in}$$\text{vivo}$ incorporation of deoxyuridine and thymidine into the DNA of rat small intestine was studied. 5-fluorouracil produced a greater than 90% inhibition of deoxyuridine incorporation within one hour after injection. This degree of inhibition was sustained for at least 12 hours. Deoxyuridine incorporation was inhibited by 30 to 65% during the initial six hours after the injection of ftorafur. By 12 hours the rate of incorporation had returned to 66% of the control value. Neither drug inhibited thymidine incorporation into DNA. A study of the metabolic disposition of radioactively labeled ftorafur and 5-fluorouracil showed that the latter drug was more rapidly and completely converted to fluorouracil-containing nucleotides in the small intestine. The possible relationship between these findings and the reported differences in the toxicity of the two drugs is discussed.     

Cloned mice from fetal fibroblast cells arrested at metaphase by a serial nuclear transfer

Cloning using G(0)-arrested somatic cells has led to the suggestion that this stage of the cell cycle is necessary for the success of cloning. In this study we report that cloned mice can be generated from fetal fibroblasts arrested at metaphase of the cell cycle. The procedure involves fusing a metaphase-arrested fetal fibroblast to an enucleated oocyte. After parthenogenetic activation a polar body and single diploid pronucleus were formed. Some of these were allowed to develop to the blastocyst stage, while others were enucleated and the nucleus was transferred to an enucleated fertilized 1-cell embryo. After the single transfer technique, 2 out of 164 developed to late stages of gestation were dead with gross abnormalities. However, after the serial nuclear transfer, 5 out of 272 embryos were recovered live at Day 19.5, and 2 of these went on to develop into apparently normal adults. All of the cloned embryos showed severe placental hypertrophy and defective differentiation of placental tissues. This study illustrates that reprogramming can occur after nuclear transfer at metaphase of the cell cycle.     

The anticlastogenic potential of fatty acid methyl esters

To test for possible anticlastogenic effects of fatty acids, the methyl esters of fatty acids--short-chain to long-chain--were examined on busulfan in Chinese hamster bone-marrow cells using the chromosome aberration test. When the experimental animals were treated with fatty acid esters and the mutagen, the chromosome-breaking actions of busulfan were not modulated by the short-chain fatty acids, but the fatty acids from lauric acid (C12) up to nonadecanoic acid (C19) reduced the rate of aberrant metaphases from 9.4 to about 3% at doses of 100 mg/kg and less. Other chemical properties of the fatty acids (saturated or not, number of double bonds, even- or odd-numbered) had no influence on the anticlastogenic effects. The only exceptions to this rule were arachidonic acid, which had no effect, and gamma-linolenic acid, which had no consistent effect on the action of busulfan.     

Developmental potential of mouse embryos reconstructed from metaphase embryonic stem cell nuclei

Mice have recently been successfully cloned from embryonic stem (ES) cells. However, these fast dividing cells provide a heterogeneous population of donor nuclei, in terms of cell cycle stage. Here we used metaphases as a source of donor nuclei because they offer the advantage of being both unambiguously recognizable and synchronous with the recipient metaphase II oocyte. We showed that metaphases from ES cells can provide a significantly higher development rate to the morula or blastocyst stage (56--70%) than interphasic nuclei (up to 28%) following injection into a recipient oocyte. Selective detachment of mitotic cells after a demecolcin treatment greatly facilitates and accelerates the reconstruction of embryos by providing a nearly pure population of cells in metaphase and did not markedly affect the developmental rate. Most of the blastocysts obtained by this procedure were normal in terms of both morphology and ratio of inner cell mass and total cell number. After transfer into pseudopregnant recipients at the one- or two-cell stage, the ability of metaphase to be fully reprogrammed was demonstrated by the birth of two pups (1.5% of activated oocytes). Although the implantation rate was quite high (up to 32.9% of activated oocytes), the postimplantation development was characterized by a high and rapid mortality. Our data provide a clear situation to explore the long-lasting effects that can be induced by early reprogramming events.     

An association between low-dose hyper-radiosensitivity and the early G2-phase checkpoint in normal fibroblasts of cancer patients

In our previous study, low-dose hyper-radiosensitivity (HRS) effect was demonstrated for normal fibroblasts (asynchronous and G2-phase enriched) of 4 of the 25 cancer patients investigated. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, the study indicated that G2-phase enrichment had no influence on HRS identification. The conclusion contradicts that reported for human tumor cells, and suggests different mechanism of HRS in normal human cells. In the present paper we report, for the first time, the activity of early G2-phase checkpoint after low-dose irradiation in normal fibroblasts of these 4 HRS-positive patients and 4 HRS-negative patients and answer the question regarding the role of this checkpoint in normal human cells. The response of the early G2-phase checkpoint was determined by assessment of the progression of irradiated cells into mitosis using the mitotic marker, phosphorylated histone H3. We found evident differences in the activity of the early G2-phase checkpoint between HRS-positive and HRS-negative fibroblasts. In HRS-positive fibroblasts the checkpoint was not triggered and DNA damage was not recognized after doses lower than 0.2 Gy resulting in HRS response. On the contrary, in HRS-negative fibroblasts the early G2-phase checkpoint was activated regardless of the dose in the range 0.1–2 Gy. In conclusion, although cell cycle phase has no effect on the presence of HRS effect in normal human fibroblasts, the data reported here indicate that HRS response in these cells is associated with the functioning of early G2-phase checkpoint in a threshold-dose dependent manner, similarly as it takes place in most of human tumor and other cells.     

MORPHOLOGICAL AND KINETIC ASPECTS OF MITOTIC ARREST BY AND RECOVERY FROM COLCEMID

The effect of Colcemid on the in vivo system of regenerating rat liver and on the in vitro system of HeLa cell cultures was studied to determine some of the morphological and kinetic aspects of metaphase blockage and recovery. The results indicated that under certain conditions the blocking effects of the drug were reversed; a functional bipolar spindle reorganized, and normal division resulted. Individual HeLa cells were followed by time-lapse cinemicrography prior to, during, and after Colcemid treatment. There was no apparent effect on cells in interphase. Cells entered mitosis at a normal rate and passed through prophase. A spindle was formed in most cells, albeit deformed, stunted, or shrunken. Within a certain range of drug concentrations, the spindle lengths showed characteristic unimodal distributions. After a 2-hr exposure to the drug followed by 1 hr in fresh medium, spindle lengths were restored to normal. Cells arrested in metaphase for periods as long as 5 hr were capable of reconstituting a normal functional spindle. Cells blocked for periods longer than 5 to 6 hr failed to recover.     

Effect of ftorafur and 5-fluorouracil on the chromosomes of human tumor cell cultures]

The effect of two antitumour drugs, ftorafur (Ft) and 5-fluorouracil (5-FU) on chromosomes of human tumour cells (strain CA-1) was studied in vitro. Since no data on the karyotype of this tumour strain had been published, the chromosome set of the model was investigated at first. Significant quantitative and structural divergence from the normal human male karyotype were observed. Steam line cells contained 47-49 chromosomes, including 9 permanent markers. No Y-chromosome was revealed. Ft and 5-FU hardly injured the chromosomes of CA-1 cells; the level of aberrant metaphases reached 94%. Chromatid deletions and gaps formed the major part of drug-induced cytogenetic abnormalities.     

Effect of cytomegalovirus on cell cycle progression and formation of pathological mitoses in cultured human diploid fibroblasts

The effect of cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G2, or M); at the same time, at least part of infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained 3H-thymidine label above chromosomes. This fact suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of cytomegalovirus.     

Contact-mediated changes in the fluidity of membrane lipids in normal and malignant transformed mammalian fibroblasts

The degree of microviscosity, gh, (fluidity/rigidity behavior) of membrane lipids of normal and transformed mammalian fibroblasts obtained from mice, hamsters and rats was quantitatively monitored by fluorescence polarization, P, analysis of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH) when embedded in lipid regions of cellular membranes of intact viable cells. Analysis of membrane microviscosity of six different cell populations and of individual cells in each cell population have indicated that the membrane microviscosity of all cell types, both normal and transformed fibroblasts, changes as a function of the cell density in the growing cultures. The membrane microviscosity was found to be low (high lipid fluidity) in sparse conditions but high (high lipid rigidity) in dense conditions. The induced changes in membrane microviscosity are practically reversible for all cell types and a complete reversion can be obtained within a few hours after changing the cell density conditions from sparse to dense and vice versa.Comparative studies with normal and transformed fibroblasts have shown that transformed fibroblasts have a more rigid lipid layer in their cellular membranes than normal or untransformed fibroblasts. The difference in membrane microviscosity between transformed and normal fibroblasts is higher in confluent conditions as compared with subconfluent cultures. These differences in the degree of fluidity of membrane lipids that are controlled by possible differences in the cellto-cell contact in normal and transformed fibroblasts may play a major role in determining the growth behavior of normal and malignant cells that are growing as a solid tissue and may have a direct effect on the control mechanisms that determine the presence or absence of the “density dependent inhibition” of growth.     

Effect of the Cytomegalovirus on Cell Cycle Progression and Pathological Mitoses in Cultured Human Diploid Fibroblasts

The effect of the cytomegalovirus on the cell cycle was studied autoradiographically in an asynchronous culture of human diploid fibroblasts. The analysis of labeled mitosis showed that some cells infected in the S phase ceased to progress through the cell cycle at one of its phases (S, G ₂, or M); at the same time, at least part of the infected cells remained capable of entering mitosis. Beginning from day 2 after infection by cytomegalovirus, the accumulation of pathological mitotic cells blocked at metaphase was observed in the culture. Approximately 50% of these cells contained ³H-thymidine label above chromosomes. This suggested the possibility of pathological mitosis in cells that were infected both at the S and other phases of the cell cycle. The detailed morphological analysis of chromosomes at different stages of infection demonstrated that the degree of their morphological changes increases from slight (stronger condensation) to severe pathology (fragmentation). In the aggregate, the results of the study suggested that abnormal chromosome morphology resulted from irreversible cell division arrest under the effect of the cytomegalovirus.     

Alterations in metaphase durations in cells derived from human tumours

With the use of time-lapse cinemicrography, we previously found that metaphase durations were significantly prolonged in SV40-transformed human fibroblasts when compared to untransformed controls. This was consistent with some earlier reports and suggested that prolonged metaphases could account for high metaphase/prophase ratios and possibly, in part, for increased mitotic indices seen in advanced tumours. However, there are inconsistencies in the literature and no comparable data available from malignant carcinomas. Presented in this paper are data from two cervical dysplasias, two cases of carcinoma in situ, nine malignant carcinomas and several other types of human cells. The results show that mean metaphase durations were prolonged in cells derived from most of the carcinomas but not from the other cell types. On the other hand, cytokinesis appears to progress more rapidly than normal in most of the tumour-derived cells. These and other findings indicate that the changes are a result of some metabolic alteration common to many but not all tumour cells. For reasons presented, we suggest as a working hypothesis that the alterations may be due to changes in calcium regulation, possibly resulting from alterations in mitochondrial metabolism.     

The chromosome-breaking activity of the restriction endonuclease Alu I in CHO cells is independent of the S-phase of the cell cycle

The restriction endonuclease Alu I induces chromosomal aberrations in living Chinese hamster ovary (CHO) cells. Multiple fixation times reveal that the chromosome-breaking activity of Alu I is similar to that of ionizing radiation in that it is independent of the S-phase of the cell cycle. These results indicate that DNA double-strand breaks are the ultimate lesions for the production of chromosomal aberrations in all stages of the cell cycle.     

Zinc depletion efficiently inhibits pancreatic cancer cell growth by increasing the ratio of antiproliferative/proliferative genes

We investigated the ability of the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to reduce pancreatic cancer cell viability. TPEN was much more efficient to inhibit pancreatic adenocarcinoma cell growth than a panel of anti-cancer drugs, including 5-fluorouracil, irinotecan, cisplatin, edelfosine, trichostatin A, mitomycin C, and gemcitabine, the gold standard chemotherapeutic agent for pancreatic cancer. Moreover, TPEN showed a dose- and time-dependent anti-proliferative effect significantly higher on pancreatic cancer cells than on normal primary fibroblasts. This effect may be explained by a significantly higher zinc depletion by TPEN in pancreatic cancer cells as compared to fibroblasts. Cell viability reduction by TPEN was associated to both G1-phase cell cycle arrest and apoptosis, and to the increased ratio of the expression level of cyclin-Cdk inhibitor versus cyclin genes and apoptotic versus anti-apoptotic genes. Finally, we show that apoptotic cell death induced by TPEN involved mitochondrial injury and caspase 3 and caspase 8 activation. In this study, we suggest that zinc depletion may be an efficient strategy in the treatment of pancreatic cancer because of its reduced antiproliferative effect on normal cells.