Cultured human keratinocytes: discrimination of different cell cycle compartments based upon measurement of nuclear RNA or total cellular RNA content

Abstract Correlated measurements of total cellular RNA and DNA of cultured human keratinocytes by flow cytometry, followed by multivariate analysis, discriminate three distinct subpopulations of cells differing in RNA content. The first subpopulation is comprised of small cells resembling basal cells of epidermis, with low RNA content and long (100–300 h) generation times. The second subpopulation consists of keratinocytes resembling cells in the spinous layer of epidermis, characterized by increased RNA content and shorter (35–40 h) generation times. The third subpopulation consists of the largest, keratinohyalin-containing cells which remain in G₁ and undergo terminal differentiation. In contrast to total cellular RNA, correlated measurements of DNA and nuclear RNA reveal that: (1) entrance of all cultured cells from G₁ into S phase occurs only after accumulation of the same, threshold amount of nuclear RNA; hence there is only a single population of S + G₂+ M-phase cells; (2) there are two distinct subpopulations in G₁, one with minimal nuclear RNA content and another with increased RNA. Stathmokinetic experiments indicate that the G₁-phase cells with low nuclear RNA have distinctly longer residence times in G₁ compared to cells with high nuclear RNA content. Thus, measurements of the total cellular RNA versus nuclear RNA content reveal kinetically distinct cell subpopulations. Whereas total cellular RNA content correlates more with differentiation, nuclear RNA content reflects primarily the kinetic properties of the cell.     

Influence of the G2 cell cycle block abrogator pentoxifylline on the expression and subcellular location of cyclin B1 and p34cdc2 in HeLa cervical carcinoma cells

The progression of cells from G₂ into mitosis is mainly controlled by formation of the cyclin B1/p34^cdc2 complex. The behaviour of this complex in the irradiation-induced G₂ cell cycle delay is still unclear. A prior study demonstrated that the expression of the cyclin B1 protein is reduced by irradiation, and restored to control levels by the methylxanthine drug pentoxifylline, which is a potent G₂ block abrogator. The present study shows that irradiation, and 2 mM pentoxifylline affect the expression of the cyclin-dependent kinase p34^cdc2 in HeLa cells. Irradiation induces p34^cdc2 levels to increase and cyclin B1 levels to decrease. Addition of pentoxifylline at the G₂ maximum reverses these trends. This is also evident from the cyclin B1/p34^cdc2 ratios which decline after irradiation and are rapidly restored to control levels upon addition of pentoxifylline. It is concluded that cyclin B1 and p34^cdc2 protein expression are important events and act in concert to control the irradiation induced G₂ block. Analysis of cyclin B1 expression in whole cells and in isolated nuclei furthermore show that cyclin B1 is translocated from the nucleus into the cytoplasm when the G₂ block is abrogated by pentoxifylline.     

Distinct functions of Nijmegen breakage syndrome in ataxia telangiectasia mutated-dependent responses to DNA damage

Phosphorylation of NBS1, the product of the gene mutated in Nijmegen breakage syndrome (NBS), by ataxia telangiectasia mutated (ATM), the product of the gene mutated in ataxia telangiectasia, is required for activation of the S phase checkpoint in response to ionizing radiation (IR). However, NBS1 is also thought to play additional roles in the cellular response to DNA damage. To clarify these additional functions of NBS1, we generated NBS cell lines stably expressing various NBS1 mutants from retroviral vectors. The ATM-dependent activation of CHK2 by IR was defective in NBS cells but was restored by ectopic expression of wild-type NBS1. The defects in ATM-dependent activation of CHK2, S phase checkpoint control, IR-induced nuclear focus formation, and radiation sensitivity apparent in NBS cells were not corrected by expression of NBS1 mutants that lack an intact MRE11 binding domain, suggesting that formation of the NBS1-MRE11-RAD50 complex is required for the corresponding normal phenotypes. Expression of NBS1 proteins with mutated ATM-targeted phosphorylation sites (serines 278 or 343) did not restore S phase checkpoint control but did restore the ability of IR to activate CHK2 and to induce nuclear focus formation and normalized the radiation sensitivity of NBS cells. Expression of NBS1 containing mutations in the forkhead-associated or BRCA1 COOH terminus domains did not correct the defects in radiation sensitivity or nuclear focus formation but did restore S phase checkpoint control in NBS cells. Together, these data demonstrate that multiple functional domains of NBS1 are required for ATM-dependent activation of CHK2, nuclear focus formation, S phase checkpoint control, and cell survival after exposure to IR.     

RESPONSE OF MOUSE BONE MARROW COLONY FORMING UNITS IN DIFFERENT STAGES OF THE CELL CYCLE TO IN VITRO INCUBATION WITH MITOMYCIN-C

A short-term in vitro method was employed to study the Mitomycin-C sensitivity of normal mouse bone marrow CFU without triggering the G₀-phase cells into the proliferative cycle. Comparison was made of the toxicities of the drug against cells in different phases of the cell cycle including G₀. Mitomycin-c killed CFU both in and out of the S-phase. No significant difference could be found between its toxicities against normal and proliferating CFU; along the exponential part of the survival curve 1·6 μg/ml concentration of the drug reduced survival to 10%. Although in the normal bone marrow only a few CFU are in the S-phase and are killed by the agent, presence of the sensitive G₀ cells produce a significant amount of non-S-phase mortality. Among the proliferating CFU population the non-S-phase lethality is less due to the absence of G₀ cells. About 75% of the S-phase cells are killed after incubation with 1 μg/ml drug; outside the S-phase, the lethality is about 40–50%. The studies indicate that the G₀ cells which are situated near the G₁-S boundary are almost as sensitive to the drug as other non-S-phase cells like G₁ cells. The clinical significance of the findings is discussed.     

Mammalian cells are not synchronized in G1-phase by starvation or inhibition: considerations of the fundamental concept of G1-phase synchronization

Synchronization of mammalian cells by starvation-refeeding or by inhibition-release are among the most commonly used techniques for division cycle analysis. An alternative analysis—in the form of a Gedanken or thought experiment—is presented, casting doubt on the utility of this synchronization method. Arresting cell growth produces a culture where all cells contain a G₁ amount of DNA. However, these cells are not arrested at a particular point in the G₁-phase. Analysis of 'G₁ arrested cells' suggests that, upon resumption of growth, the cells are not synchronized.     

Role of protein kinase C-iota in transformed non-malignant RWPE-1 cells and androgen-independent prostate carcinoma DU-145 cells

Prostate cancer is one of the leading causes of death among men in the USA.
Objective:  In this study, we investigated the role of atypical protein kinase C-iota (PKC-ι) in androgen-independent prostate DU-145 carcinoma cells compared to transformed non-malignant prostate RWPE-1 cells.
Materials and methods:  Western blotting and immunoprecipitations demonstrated that PKC-ι is associated with cyclin-dependent kinase activating kinase (CAK/Cdk7) in RWPE-1 cells, but not in DU-145 cells.
Results:  Treatment of prostate RWPE-1 cells with PKC-ι silencing RNA (siRNA) decreased cell viability, cell-cycle accumulation at G₂/M phase, and phosphorylation of Cdk7 and Cdk2. In addition, PKC-ι siRNA treatment caused less phosphorylation of Bad at ser-155, ser-136, and greater Bad/Bcl-x_L heterodimerization, leading to apoptosis. In DU-145 cells, PKC-ι was anti-apoptotic and was required for cell survival. Treatment with PKC-ι siRNA blocked increase in cell number, and inhibited G₁/S transition by accumulation of cells in G₀/G₁ phase. In addition to cell-cycle arrest, both RWPE-1 and DU-145 cells underwent apoptosis due to mitochondrial dysfunction and apoptosis cascades, such as release of cytochrome c, activation of caspase-7, and poly (ADP-ribose) polymerase (PARP) cleavage.
Conclusion:  Our results suggest that PKC-ι is required for cell survival in both transformed non-malignant prostate RWPE-1 cells and androgen-independent malignant prostate DU-145 cells, whereas suppressing PKC-ι lead to apoptosis in DU-145 prostate cells.     

Effect of separase depletion on ionizing radiation-induced cell cycle checkpoints and survival in human lung cancer cell lines

Abstract.   Objectives : This study is to evaluate the effect of separase depletion on cell cycle progression of irradiated and non-irradiated cells through the G₂/M phases and consecutive cell survival. Materials and methods : Separase was depleted with siRNA in two human non-small cell lung carcinoma (NSCLC) cell lines. Cell cycle progression, mitotic fraction, DNA repair, apoptotic and clonogenic cell death were determined. Results : By depletion of endogenous separase with siRNA in NSCLCs, we showed that separase affects progression through the G₂ phase. In non-irradiated exponentially growing cells, separase depletion led to an increased G₂ accumulation from 17.2% to 29.1% in H460 and from 15.7% to 30.9% in A549 cells and a decrease in mitotic cells. Depletion of separase significantly ( P <  0.01) increased the fraction of radiation-induced G₂ arrested cells 30–56 h after irradiation and led to decrease in the mitotic fraction. This was associated with increased double-strand break repair as measured by γ-H2AX foci kinetics in H460 cells and to a lesser extent in A549 cells. In addition, a decrease in the expression of mitotic linked cell death after irradiation was found. Conclusions : These results indicate that separase has additional targets involved in regulation of G₂ to M progression after DNA damage. Prolonged G₂ phase arrest in the absence of separase has consequences on repair of damaged DNA and cell death.     

Differential Coupling of μ-, δ-, and κ-Opioid Receptors to Gα16-Mediated Stimulation of Phospholipase C

Abstract: The μ-opioid receptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G₁₆ protein. Given the promiscuous nature of G₁₆ and the high degree of resemblance of signaling properties of the three opioid receptors, both δ- and κ-opioid receptors are likely to activate G₁₆. Interactions of δ- and κ-opioid receptors with G₁₆ were examined by coexpressing the opioid receptors and Gα₁₆ in COS-7 cells. The δ-selective agonist [ d -Pen², d -Pen⁵]enkephalin potently stimulated the formation of inositol phosphates in cells coexpressing the δ-opioid receptor and Gα₁₆. The δ-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of simeter for quality control of blood units and irradiators. 13.   Transfusion 1993 ; 33 : 898 – 901 . [PubMed link] 14.   Butson MJ , Yu PK , Cheung T , et al . Dosimetry of blood irradiation with radiochromic film. Transfus Med 1999 ; 9 : 205 – 8 . [PubMed link] 15.   Nath R , Biggs PJ , Ling CC , et al . AAPM code of practice for radiotherapy accelerators: Report of AAPM Radiation Therapy Task Group No. 45. Med Phys     

Interaction of FANCD2 and NBS1 in the DNA damage response

Fanconi anaemia (FA) and Nijmegen breakage syndrome (NBS) are autosomal recessive chromosome instability syndromes with distinct clinical phenotypes. Cells from individuals affected with FA are hypersensitive to mitomycin C (MMC), and cells from those with NBS are hypersensitive to ionizing radiation. Here we report that both NBS cell lines and individuals with NBS are hypersensitive to MMC, indicating that there may be functional linkage between FA and NBS. In wild-type cells, MMC activates the colocalization of the FA subtype D2 protein (FANCD2) and NBS1 protein in subnuclear foci. Ionizing radiation activates the ataxia telangiectasia kinase (ATM)-dependent and NBS1-dependent phosphorylation of FANCD2, resulting in an S-phase checkpoint. NBS1 and FANCD2 therefore cooperate in two distinct cellular functions, one involved in the DNA crosslink response and one involved in the S-phase checkpoint response.     

Nbs1 is required for ATR-dependent phosphorylation events

Nijmegen breakage syndrome (NBS) is characterised by microcephaly, developmental delay, characteristic facial features, immunodeficiency and radiosensitivity. Nbs1, the protein defective in NBS, functions in ataxia telangiectasia mutated protein (ATM)-dependent signalling likely facilitating ATM phosphorylation events. While NBS shares overlapping characteristics with ataxia telangiectasia, it also has features overlapping with ATR-Seckel (ATR: ataxia-telangiectasia and Rad3-related protein) syndrome, a subclass of Seckel syndrome mutated in ATR. We show that Nbs1 also facilitates ATR-dependent phosphorylation. NBS cell lines show a similar defect in ATR phosphorylation of Chk1, c-jun and p-53 in response to UV irradiation- and hydroxyurea (HU)-induced replication stalling. They are also impaired in ubiquitination of FANCD2 after HU treatment, which is ATR dependent. Following HU-induced replication arrest, NBS and ATR-Seckel cells show similarly impaired G2/M checkpoint arrest and an impaired ability to restart DNA synthesis at stalled replication forks. Moreover, NBS cells fail to retain ATR in the nucleus following HU treatment and extraction. Our findings suggest that Nbs1 functions in both ATR- and ATM-dependent signalling. We propose that the NBS clinical features represent the result of these combined defects.     

Radiation, DNA damage and cancer

The characterization of the rare, radiation-sensitive and cancer-prone syndromes, ataxia telangiectasia and Nijmegen breakage syndrome, has demonstrated that genetic predisposition increases the risk of developing cancer after exposure to ionizing radiation (IR). Molecular analyses of these disorders provide valuable insights into the normal function of these two gene products in the cellular response to IR-induced DNA damage. Their contribution to a cellular radiosensitive phenotype and their role in sporadic cancers can now be fully assessed. For example, the gene ataxia telangiectasia mutated (ATM) has recently been shown to be a tumour suppressor gene in T-cell prolymphocytic leukaemia, and there is increasing evidence that individuals with one mutated ATM or Nijmegen breakage syndrome (NBS1) allele have an increased predisposition to cancer.     

Cell-cycle dependence of a ganglioside glycosyltransferase activity and its inhibition by enkephalin in a neurotumor cell line

Abstract: Rat glioma mouse neuroblastoma hybrid neurotumor cells (NG108-15), synchronized by amino acid deprivation, showed a cell-cycle-dependent peak of activity of a ganglioside N-acetylgalactosaminyl transferase 14-24 h following release from the cell cycle block (S/G₂ phase). Maximal expression of two typical lysosomal hydrolases, N-acetyl-β-hexosaminidase and β-galactosidase, occurred between 18 and 21 h following release (S phase), declining to G₁ phase levels during the peak of N-acetylgalactosamine (GalNAc) transferase activity. In addition, glycosyltransferase activity in G₂ phase cells showed an increase in apparent V_max (suggesting the presence of more enzyme/mg of cell protein) and apparent binding affinity for uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) (32 versus 14 M) when compared to transferase activity in the G₁ phase. However, the opioid peptide enkephalin [D-Ala², o-Leu⁵], which inhibits ganglioside GalNAc transferase activity in unsynchronized NG108-15 cultures, was much more inhibitory in whole cells 8 h after release from the cell cycle block (G₁ phase) than in cells 20 h after release (G, phase), with 50% inhibition occurring at 2 ± 10^-9M and 2 ± 10^-7M, respectively. These results suggest that the GalNAc transferase activity is regulated in more than one way during the cell cycle, since both V_max and K_m changes are observed, and that the cyclic AMP-dependent mechanism by which opiates reduce transferase activity is receptor mediated and cell cycle dependent.     

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON THE CELL PROGRESS FROM M TO G1 AND S STAGES IN CULTURED MOUSE LEUKEMIA L5178Y CELLS

Actinomycin D (0.5 μg/ml) did not prevent M stage cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The position of the block was approximately 1.4 hr before S stage or just after the beginning of G₁ stage. Actinomycin D in this concentration also significantly depressed uridine-³H uptake into G₁ stage cells, but did not suppress leucine-³H uptake by M and G₁ cells. This suggests that some proteins may be synthesized in M and G₁ stage cells by messenger RNA left over from the previous cell cycle. However, entry of G₁ cells into S stage would require synthesis of new messenger RNA near the beginning of G₁ stage. Puromycin (10 μg/ml) did not prevent M cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The site of blockage was about 0.7 hr before S stage or in the first two-third of G₁ stage. This might be the site where the cells synthesize new G₁ proteins necessary for entry to S stage.
Comparison of sensitivities of G₁ and G₂ stages to the two antibiotics reveals that the puromycin sensitivity of G₁ cells was similar to that of G₂ cells, but the actinomycin D sensitivity of G₁ was greater than that of G₂ cells.     

Is there an accumulation of cells during G2 in some acute lymphoblastic leukaemias?

Abstract. In some cases of acute lymphoblastic leukaemia (ALL) the percentage of cells in G₂+ M is higher than anticipated when compared with the percentage in S phase. This increase in G₂+ M, as detected by flow cytometry measurement of DNA content, may be due to an accumulation of cells, either in G ₂ or during the end of S phase; it may also be related to the existence of small tetraploid clones generally ignored by cytogeneticists. In order to identify possible subpopulations of cells with a DNA index ≥ 2-0, we have compared the results of a cytogenetic analysis to the G₂+ M values. We have also studied the distribution of S phase cells in 24 cases of ALL by incorporating 5-bromodeoxyuridine, labelling the cells by indirect immunofluorescence, and analysing them by flow cytometry after propidium iodide staining. The distribution of cells during S phase was quantified: no accumulation of cells was ever observed at the end of S phase. The question of the existence of small tetraploid clones, G₂ arrested cells or cells with a G₂ elongation remains open. However, we feel that it is more probable that, in this pathology, an elongation of the duration of G₂ occurs.     

ON THE RESTING STAGES OF THE JB-1 ASCITES TUMOUR

Cytophotometric determination of single-cell DNA after repeated ³H-thymidine labelling of the JB-1 ascites tumour in the plateau phase of growth showed a massive accumulation of unlabelled cells with both G₁ and G₂ content. Autoradiography combined with cytophotometry or colcemid block demonstrated that some of these unlabelled cells were rapidly triggered into the cell cycle when plateau tumours were transferred to new hosts. This indicated that tumour cells may be held up in non-cycling stages corresponding to both the G₁ and the G₂ phase of the cell cycle.     

Method For Probing Cells In Radiation-Induced G2Arrest: Demonstration of Potentially Lethal Damage Repair

Abstract. Chinese hamster ovary cells were arrested in the G₂ phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G₂, for the investigation of G₂ arrest. Comparisons were made of the number of cells retrieved from G₂ arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. the retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. the repair curves (surviving fraction us time) were each described by a single exponential. G₂ cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

High temperature causes arrest of cell cycle in G2 phase in BmN cells derived from the silkworm, Bombyx mori

Abstract.  The influence of temperature on the insect cell line, BmN, derived from the silkworm, Bombyx mori is investigated. These cells proliferate at an accelerated pace as the temperature increases from 22 to 30 °C, but the growth rate slows at 34 °C, and proliferation stops at 38 °C. At high temperatures, abnormal cellular morphology is observed. Cells treated at 38 °C have cytoplasmic bilateral protrusions and they gradually aggregate and float in the medium. BmN cells without proliferation at 38 °C are viable but have reduced DNA synthesis. At high temperatures, the cell cycle of BmN cells halts at the G₂ phase. After heat treatment of the larvae, an accumulation of larval haemocytes with high DNA content is found, which suggests that the cell cycle arrest at G₂ also occurs in the silkworm at high temperatures.