Biomedical subjects

T Stokke

Publications and source records attributed to T Stokke.

At least 37 records · Page 2Linked to original sources

PubMed1999

Prognostic value of lymphoma-specific S-phase fraction compared with that of other cell proliferation markers.

The proliferation-associated antigens Ki67 (immunohistochemistry) and proliferative cell nuclear antigen (PCNA) (immunohistochemistry and immunoblotting) were analysed together with DNA synthesis (3H-thymidine incorporation) and cell-cycle distribution (tumour-specific S-phase fraction determined by flow cytometry) in lymph node suspensions from 63 patients with newly diagnosed B-Cell non-Hodgkin's lymphomas. Details of clinical parameters, treatment and patient outcome were available for all patients, and retrospectively analysed. Of the proliferation-associated parameters, only high S-phase fraction (p < 0.00001) and high PCNA expression by immunoblotting (p = 0.012) were predictive of a poor prognosis. Of the conventional parameters, high-grade malignancy, high International Prognostic Index (IPI) score, bulky disease and presence of B symptoms predicted a patient for poor survival. High S-phase fraction was predictive of a short survival for the low-grade lymphomas analysed separately (p < 0.00001), as well as for patients treated with an Adriamycin- and a non-Adriamycin-containing regimen (p < 0.005 for both groups). In a multivariate analysis, S-phase fraction (p = 0.00006), IPI score (p = 0.015) and B symptoms (p = 0.017) had independent prognostic values, but not histological grade.

Adult↗

PubMed1999

Regulation of the start of DNA replication in Schizosaccharomyces pombe.

Cells of Schizosaccharomyces pombe were grown in minimal medium with different nitrogen sources under steady-state conditions, with doubling times ranging from 2.5 to 14 hours. Flow cytometry and fluorescence microscopy confirmed earlier findings that at rapid growth rates, the G1 phase was short and cell separation occurred at the end of S phase. For some nitrogen sources, the growth rate was greatly decreased, the G1 phase occupied 30-50% of the cell cycle, and cell separation occurred in early G1. In contrast, other nitrogen sources supported low growth rates without any significant increase in G1 duration. The method described allows manipulation of the length of G1 and the relative cell cycle position of S phase in wild-type cells. Cell mass was measured by flow cytometry as scattered light and as protein-associated fluorescence. The extensions of G1 were not related to cell mass at entry into S phase. Our data do not support the hypothesis that the cells must reach a certain fixed, critical mass before entry into S. We suggest that cell mass at the G1/S transition point is variable and determined by a set of molecular parameters. In the present experiments, these parameters were influenced by the different nitrogen sources in a way that was independent of the actual growth rate.

Biomass↗

PubMed1998

Propidium iodide quenches the fluorescence of TdT-incorporated FITC-labeled dUTP in apoptotic cells.

Apoptotic cells with frequent DNA strand breaks may be detected by tagging with directly or indirectly labeled nucleotides incorporated by the use of terminal deoxynucleotidyl transferase (TdT). Propidium iodide (PI) is typically added for the simultaneous assessment of DNA content. PI was found to quench the specific in situ FITC-fluorescence of apoptotic cells which were labeled by TdT with FITC-conjugated dUTP, biotin-dUTP followed by streptavidin-FITC, or digoxigenin-dUTP followed by FITC-labeled anti-digoxigenin antibodies as measured by flow cytometry. The effect was concentration-dependent, with 50% quenching occurring at 0.8 microg/ml, 1.5 microg/ml, and 5 microg/ml PI, respectively, at approximately 1 x 10(6) cells/ml. Spectrofluorimetry in solution revealed that 15 microg/ml PI was required to quench 50% of the fluorescence of ss FITC-labeled poly(dU)35. In contrast, the fluorescence of ds FITC-labeled poly(dU)35-poly(dA) was quenched to 50% at 3 microg/ml PI. The maximum of the fluorescence excitation spectrum of PI shifted from 490 nm to 535 nm upon binding to ds DNA as well as ss poly(dU)35, and the fluorescence yield of PI at 610 nm increased, but the binding required 10-fold higher concentrations of poly(dU)35 as compared to ds DNA. The spectroscopic properties of PI are therefore similar whether bound to poly(dU) or to double-stranded DNA, but the binding to poly(dU) is much weaker. The observed quenching in situ therefore cannot be explained by direct binding of PI to the poly(dU) tails synthesized by TdT in situ in apoptotic cells, but may rather be due to radiationless energy transfer from FITC to PI bound to double-stranded DNA close to the nicks where TdT is known to start polymerization.

Apoptosis↗

PubMed1998

Protoporphyrin IX accumulation in cells treated with 5-aminolevulinic acid: dependence on cell density, cell size and cell cycle.

Human colon adenocarcinoma cells (WiDr) and Chinese hamster lung fibroblasts cells (V79) were incubated with different concentrations of 5-aminolevulinic acid (ALA), and the production of protoporphyrin IX (PpIX) was studied using several techniques. The amount of PpIX produced per cell increased with increasing ALA concentration according to different kinetics for the 2 cell lines. For both cell lines a cell density dependency of the PpIX synthesis was observed. For saturating ALA concentrations, 2-3 times more PpIX was produced per cell at a density of 5 x 10(4) than at a density of 5 x 10(3) cells/cm2. The photosensitivity of cells appeared to increase even more than the PpIX content, indicating a cooperative effect in inactivation. The PpIX production rate increased with cell size and was about 1.9 times higher for cells in the G2 + M phase than for cells in the G1 phase of the cell cycle. Neither cell size nor cell cycle distribution were significantly dependent on cell density.

Adenocarcinoma↗

PubMed1998

Flow cytometry of bacterial cells: comparison between different flow cytometers and different DNA stains.

The DNA content and light scatter from individual Escherichia coli cells were measured in two flow cytometers with different configurations. The DNA content could be measured with similar resolution either in an Argus flow cytometer equipped with a mercury lamp, or in a FACStar flow cytometer with two argon lasers as light sources. In contrast, light scatter measurements appeared to be a good measure of cell mass only in the Argus instrument. Three DNA stains were compared:, DAPI, Hoechst 33258, and mithramycin A together with ethidium bromide. All three stains yielded DNA histograms of similar quality in both flow cytometers. Optimal results required that the stain and cell concentrations were kept similar, that a fixed rate of sample introduction was used, and that a period of equilibration was allowed during running of each sample. The results demonstrate that conventional, laser-based flow cytometers may be used for high-resolution measurements of bacterial DNA content, thereby making flow cytometry available to an increased number of research groups working with prokaryotes.

Cell Separation↗

PubMed1998

The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein.

We have studied the role of the oxygen-dependent pyrimidine metabolism in the regulation of cell cycle progression under moderate hypoxia in human cell lines containing functional (T-47D) or non-functional (NHIK 3025, SAOS-2) retinoblastoma gene product (pRB). Under aerobic conditions, pRB exerts its growth-regulatory effects during early G1 phase of the cell cycle, when all pRB present has been assumed to be in the underphosphorylated form and bound in the nucleus. We demonstrate that pRB is dephosphorylated and re-bound in the nucleus in approximately 90% of T-47D cells located in S and G2 phases under moderately hypoxic conditions. Under these conditions, no T-47D cells entered S-phase, and no progression through S-phase was observed. Progression of cells through G2 and mitosis seems independent of their functional pRB status. The p21WAF1/CIP1 protein level was significantly reduced by moderate hypoxia in p53-deficient T-47D cells, whereas p16(INK4a) was not expressed in these cells, suggesting that the hypoxia-induced cell cycle arrest is independent of these cyclin-dependent kinase inhibitors. The addition of pyrimidine deoxynucleosides did not release T-47D cells, containing mainly underphosphorylated pRB, from the cell cycle arrest induced by moderate hypoxia. However, NHIK 3025 cells, in which pRB is abrogated by expression of the HPV18 E7 oncoprotein, and SAOS-2 cells, which lack pRB expression, continued cell cycle progression under moderate hypoxia provided that excess pyrimidine deoxynucleosides were present. NHIK 3025 cells express high levels of p16INK4a under both aerobic and moderately hypoxic conditions, suggesting that the inhibitory function of p16(INK4a) would not be manifested in such pRB-deficient cells. Thus, pRB, a key member of the cell cycle checkpoint network, seems to play a major role by inducing growth arrest under moderate hypoxia, and it gradually overrides hypoxia-induced suppression of pyrimidine metabolism in the regulation of progression through S-phase under such conditions.

Aerobiosis↗

PubMed1998

Proliferation and apoptosis in malignant and normal cells in B-cell non-Hodgkin's lymphomas.

We have examined apoptosis and proliferation in lymph node cell suspensions from patients with B-cell non-Hodgkin's lymphoma using flow cytometry. A method was developed which allowed estimation of the fractions of apoptotic cells and cells in the S-phase of the cell cycle simultaneously with tumour-characteristic light chain expression. Analysis of the tumour S-phase fraction and the tumour apoptotic fraction in lymph node cell suspensions from 95 B-cell non-Hodgkin's lymphoma (NHL) patients revealed a non-normal distribution for both parameters. The median fraction of apoptotic tumour cells was 1.1% (25 percentiles 0.5%, 2.7%). In the same samples, the median fraction of apoptotic normal cells was higher than for the tumour cells (1.9%; 25 percentiles 0.7%, 4.0%; P = 0.03). The median fraction of tumour cells in S-phase was 1.4% (25 percentiles 0.8%, 4.8%), the median fraction of normal cells in S-phase was significantly lower than for the tumour cells (1.0%; 25 percentiles 0.6%, 1.9%; P = 0.004). When the number of cases was plotted against the logarithm of the S-phase fraction of the tumour cells, a distribution with two Gaussian peaks was needed to fit the data. One peak was centred around an S-phase fraction of 0.9%; the other was centred around 7%. These peaks were separated by a valley at approximately 3%, indicating that the S-phase fraction in NHL can be classified as 'low' (< 3%) or 'high' (> 3%), independent of the median S-phase fraction. The apoptotic fractions were log-normally distributed. The median apoptotic fraction was higher (1.5%) in the 'high' S-phase group than in the 'low' S-phase group (0.8%; P = 0.02). However, there was no significant correlation between the two parameters (P > 0.05).

Adult↗

PubMed1998

Tumour cell proliferation, but not apoptosis, predicts survival in B-cell non-Hodgkin's lymphomas.

Tumour S-phase fraction, but not the apoptotic fraction, had prognostic value in 92 patients with B-cell non-Hodgkin's lymphoma (P < 0.0001 and P = 0.85 respectively). Multivariate analysis showed that S-phase fraction was the strongest prognostic indicator in all cases (P = 0.0003, relative risk 4.3; age: P = 0.16; grade: P = 0.81), as well as in the 63 primary biopsy cases (P = 0.0006, relative risk = 7.3; international prognostic index: P = 0.015, relative risk = 3.2; B symptoms: P = 0.017, relative risk = 3.3; bulkiness: P = 0.65; grade: P = 0.91).

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1998

Hypoxia induces p53 accumulation in the S-phase and accumulation of hypophosphorylated retinoblastoma protein in all cell cycle phases of human melanoma cells.

Hypoxia has been shown to induce accumulation of p53 and of hypophosphorylated retinoblastoma protein (pRb) in tumour cells. In this study, the cell cycle dependence of p53 accumulation and pRb hypophosphorylation in four human melanoma cell lines that are wild type for p53 was investigated using two-parameter flow cytometry measurements of p53 or pRb protein content and DNA content. The hypoxia-induced increase in p53 protein was higher in S-phase than in G1 and G2 phases in all cell lines. The accumulation of p53 in S-phase during hypoxia was not related to hypoxia-induced apoptosis or substantial cell cycle specific cell inactivation during the first 24 h of reoxygenation. pRb was hypophosphorylated in all cell cycle phases by hypoxia treatment. The results did not support a direct link between p53 and pRb during hypoxia because p53 was induced in a cell cycle-specific manner, whereas no cell cycle-dependent differences in pRb hypophosphorylation were detected. Only a fraction of the cell populations (0.60+/-0.10) showed hypophosphorylated pRb. Thus, pRb is probably not the only mediator of the hypoxia-induced cell cycle block seen in all cells and all cell cycle phases. Moreover, the cell cycle-dependent induction of p53 by hypoxia suggests that the primary function of p53 accumulation during hypoxia is other than to arrest the cells.

Apoptosis↗

PubMed1998

Apoptosis and expression of Bax, Bcl-x, and Bcl-2 apoptotic regulatory proteins in colorectal carcinomas, and association with p53 genotype/phenotype.

AIMS: Spontaneous apoptosis and expression of the apoptotic regulatory proteins Bax, Bcl-x, and Bcl-2 were investigated in 50 colorectal carcinomas. The p53 genotypes/phenotypes and BAX genotypes were also determined, and possible associations of these with apoptosis and/or with expression of the different apoptotic regulatory proteins were studied. METHODS: Terminal deoxynucleotidyl transferase (TdT) mediated dUTP labelling of DNA fragments was used to detect apoptotic tumour cells in sections and peroxidase immunohistochemistry was used to assess protein expression. p53 genotype/phenotype was determined using constant denaturant gel electrophoresis/immunoblotting and bax genotype was determined using polymerase chain reaction based methods. RESULTS: The distribution of tumour apoptotic indices was bimodal with a natural cut off at 1.0% (range, 0.0-5.4%); the median fraction of apoptotic tumour cells was 0.8%. Tumour apoptosis was not associated significantly with tumour DNA ploidy status. Normal mucosal tissue had less than 0.1% apoptotic cells. Staining intensities for Bax, Bcl-x, and Bcl-2 were strong; that is, equivalent to or greater than positive normal mucosal cells, in 11 of 50, 20 of 49, and 20 of 48 carcinomas. Frameshift mutations in the bax gene were detected in three of 42 tumours analysed, all of which were DNA diploid, and Bax protein expression in these tumours was absent or very low. Bax, Bcl-x, and Bcl-2 protein expression were not correlated with tumour apoptosis or tumour DNA ploidy status. p53 was expressed in 34 of 50 tumours and p53 gene mutations were detected in 22 of 29 p53 positive tumours analysed. Apoptosis was significantly lower in a greater number of p53 positive tumours than p53 negative tumours. In addition, Bcl-2 protein expression was significantly higher in a greater number of p53 positive tumours compared with p53 negative tumours. Bax and Bcl-x protein expression were not significantly associated with p53 phenotype/genotype. CONCLUSIONS: The results indicate that acquisition of a p53 phenotype is associated with lower spontaneous apoptosis and higher expression of Bcl-2. The results also suggest that p53 is not a major determinant for Bax expression in colorectal carcinomas in vivo.

Apoptosis↗

PubMed1997

Hypoxia-induced apoptosis in human cells with normal p53 status and function, without any alteration in the nuclear protein level.

We have studied hypoxia-induced inactivation of cells from three established human cell lines with different p53 status. Hypoxia was found to induce apoptosis in cells expressing wild-type p53 (MCF-7 cells), but not in cells where p53 is either mutated (T-47D cells), or abrogated by expression of the HPV18 E6 oncoprotein (NHIK 3025 cells). Apoptosis was demonstrated by DNA fragmentation, using agarose gel electrophoresis of DNA and DNA nick end labeling (TUNEL). We demonstrate that extremely hypoxic conditions (< 4 ppm O2) do not cause any change of expression in the p53 protein level in these three cell lines. In addition, the localization of p53 in MCF-7 cells was found exclusively in the nucleus in only some of the cells both under aerobic and hypoxic conditions. Furthermore, no correlation was found between the p53-expression level and whether or not a cell underwent apoptosis. Flow cytometric TUNEL analysis of MCF-7 cells revealed that initiation of apoptosis occurred in all phases of the cell cycle, although predominantly for cells in S phase. Apoptosis was observed only during a limited time window (i.e., approximately 10 to approximately 24 h) after the onset of extreme hypoxia. While 66% of the MCF-7 cells lost their ability to form visible colonies following 15 h exposure to extreme hypoxia, only approximately 28% were induced to apoptosis, suggesting that approximately 38% were inactivated by other death processes. Commitment to apoptotic cell death was observed in MCF-7 cells even for oxygen concentrations as high as 5000 ppm. Our present results indicate that the p53 status in these three tumor cell lines does not have any major influence on cell's survival following exposure to extremely hypoxic conditions, whereas following moderate hypoxia, cells expressing functional p53 enhanced their susceptibility to cell death. Taken together, although these results suggest that functional p53 might play a role in the induction of apoptosis during hypoxia, other factors seem to be equally important.

Aerobiosis↗

PubMed1997

Measurement of nuclear DNA content in fission yeast by flow cytometry.

Cell division cycle (cdc) mutants of Schizosaccharomyces pombe are arrested at specific points in the cell cycle when grown at restrictive temperature. Flow cytometry of such cells reveals an anomalous increase in the DNA fluorescence signal, which represents a problem in experiments designed to determine the cell cycle arrest point. The increased fluorescence signal is due to cytoplasmic constituents and has been attributed to mitochondrial DNA synthesis (S. Sazer and S. W. Sherwood, J. Cell Sci. 97: 509-516, 1990). Here we have studied the cdc10 mutant by flow cytometry using different DNA-binding fluorochromes and found no evidence that the increased fluorescence signal was caused by mitochondrial DNA synthesis. To determine more accurately the nuclear DNA content we have developed a novel method to remove most of the cytoplasmic material by exposing the cells to Triton X-100 and hypotonic conditions after cell wall digestion. The DNA fluorescence from cells treated in this way was more constant with time of incubation at restrictive temperature in spite of a considerable increase in cell size. With this method we could determine that the recently isolated temperature sensitive orp1 mutant is arrested with a 1C DNA content. Premature and abnormal mitosis ('cut') could be observed for the orp1 mutant after only 4 h at restrictive temperature.

Cell Cycle Proteins↗

PubMed1997

A comparison of different modes for the detection of p53 protein accumulation. A study of bladder cancer.

The aim of the present study was to evaluate different techniques for the analysis of p53 protein accumulation in human bladder cancer. The accumulation was evaluated in 23 carcinomas by immunoblotting (IB), immunohistochemistry (IHC) and flow cytometry (FCM). The results revealed that six (26%), eight (35%) and ten (43%) of the tumours were p53 protein positive by IB, IHC and FCM, respectively. Mutation analysis of the TP53 gene confirmed mutations in 8 of 9 tumours which showed increased levels of p53 protein by FCM. Our results indicate that IHC could be applied for studies of p53 protein accumulation in archival formalin fixed, paraffin-embedded bladder tumours. However, FCM is a more sensitive and objective method for the detection of p53 protein than IHC and this should be taken into account when routinely evaluating the p53 protein accumulation by IHC.

Biopsy↗

PubMed1997

NO38 expression and nucleolar counts are correlated with cellular DNA content but not with proliferation parameters in colorectal carcinomas.

AIMS: To investigate the expression of nucleolar protein NO38, to determine the numbers of nucleoli per cell, and to examine the relations of these nucleolar parameters to tumour DNA index, total cellular DNA content, S phase fraction, and Ki67 labelling index. METHODS: 36 colorectal tumours and 14 normal mucosas were studied. An anti-NO38 monoclonal antibody, 31A12, and flow cytometric analysis were used to detect expression of NO38 by means of a biotin-streptavidin-FITC (fluorescein isothiocyanate) staining method. Nucleolar counts were determined using fluorescence microscopy. Flow cytometry was used to determine tumour DNA indices and the sizes of the S phase fractions. Ki67 labelling indices were determined from tissue sections stained immunohistochemically with the MIB-1 antibody against the Ki67 nuclear protein. RESULTS: Generally, tumour cell nucleoli were larger and more irregular in shape compared with nucleoli in normal mucosal cells. DNA aneuploid and diploid tumours expressed 2.8 and 2.1 times more NO38 than normal mucosa. The mean (SD) values for nucleolar counts were higher for the DNA aneuploid tumours (3.81 (0.93)) than the diploid tumours (2.62 (0.38)) and normal mucosa (2.34 (0.37)). NO38 expression and numbers of nucleoli correlated significantly (r = 0.52, p = 0.01). There were, however, no significant correlations between these nucleolar parameters and either the sizes of tumour S phase fractions or Ki67 labelling indices. Cell cycle resolved expression of NO38 in tumours and normal mucosa demonstrated that expression increased approximately in proportion to the DNA content throughout the cell cycle. In aneuploid tumours, NO38 expression was 43% and 98% higher in S and G2 phases, respectively, compared with the G1 phase. Sorting of these populations revealed that the nucleolar count also increased as the DNA content increased but by only 29% and 47% in S and G2, respectively. Apoptotic cells lacked NO38. CONCLUSIONS: NO38 expression is higher in tumours than in normal mucosa owing to the increased DNA content and larger nucleoli in tumours; expression increases proportionally with DNA content as cells progress through the cell cycle from G1 through S and G2. However, NO38 expression does not correlate with the tumour S phase fraction or Ki67 labelling index and is lost during apoptosis. Also the results suggest that nucleoli grow in size during the cell cycle, which would account for the doubling of NO38 expression from G1 to G2, as the nucleolar count increased by only 47%.

Cell Division↗

PubMed1997

Uncoupling of the order of the S and M phases: effects of staurosporine on human cell cycle kinases.

The protein kinase inhibitor staurosporine (SSP) was employed to study the involvement of kinases in human cell cycle progression. Thirty to 100 ng/ml SSP blocks entry into S phase and M phase. Lack of entry into S phase is due to impaired activity of the retinoblastoma protein kinase. The requirement for any of the SSP-sensitive kinases for cell cycle progression can be abrogated in tumour cells. Therefore, these kinases act in a checkpoint network negatively controlling the initiation of S phase, M phase and cytokinesis, rather than being inherent parts of a substrate-product chain required for the initiation of the cell cycle phases. As a consequence of the lack of certain checkpoint effectors, tumour cells may endoreduplicate or binucleate in the presence of SSP. The latter processes, as well as meiosis, are naturally occurring in specialized cell types, leading to the idea that this checkpoint network controls the order of the cell cycle phases in normal cells. A model is presented where the cell cycle is envisioned as two independently running cycles, the S and the M cycle, which are controlled by intra and intercycledependent checkpoints in human somatic cells. The model accounts for the dependency of S and M phase initiation on the successful completion of the previous M and S phase, respectively, as well as entry into a resting state.

Cell Cycle↗

PubMed1996

Coordinating DNA replication initiation with cell growth: differential roles for DnaA and SeqA proteins.

We describe here the development of a new approach to the analysis of Escherichia coli replication control. Cells were grown at low growth rates, in which case the bacterial cell cycle approximates that of eukaryotic cells with G1, S, and G2 phases: cell division is followed sequentially by a gap period without DNA replication, replication of the single chromosome, another gap period, and finally the next cell division. Flow cytometry of such slowly growing cells reveals the timing of replication initiation as a function of cell mass. The data show that initiation is normally coupled to cell physiology extremely tightly: the distribution of individual cell masses at the time of initiation in wild-type cells is very narrow, with a coefficient of variation of less than 9%. Furthermore, a comparison between wild-type and seqA mutant cells shows that initiation occurs at a 10-20% lower mass in the seqA mutant, providing direct evidence that SeqA is a bona fide negative regulator of replication initiation. In dnaA (Ts) mutants the opposite is found: the mass at initiation is dramatically increased and the variability in cell mass at initiation is much higher than that for wild-type cells. In contrast to wild-type and dnaA(Ts) cells, seqA mutant cells frequently go through two initiation events per cell division cycle, and all the origins present in each cell are not initiated in synchrony. The implications for the complex interplay amongst growth, cell division, and DNA replication are discussed.

Bacterial Outer Membrane Proteins↗

PubMed1996

The retinoblastoma gene product is reversibly dephosphorylated and bound in the nucleus in S and G2 phases during hypoxic stress.

We have studied the role of the retinoblastoma susceptibility gene product (pRB) in the regulation of cell-cycle progression under extremely hypoxic conditions (< 4 ppm O2). pRB is a nuclear matrix-associated phosphoprotein that normally exerts its growth-regulatory effects during early-G1 phase of the cell cycle, where all pRB present has been assumed to be in the under-phosphorylated form and bound in the nucleus. The effect of hypoxia on pRB nuclear binding and its state of phosphorylation was studied by two methods: (a) two-parametric flow cytometric measurement of pRB versus DNA and (b) Western blotting. Pulse-chase and pulse labeling with BrdUrd was used to record cell-cycle progression under versus after extremely hypoxic conditions. We demonstrate that pRB is dephosphorylated and rebound in the nucleus in more than 90% of cells located in S and G2 under extremely hypoxic conditions. While inhibition of DNA synthesis was instantaneous under hypoxic conditions, dephosphorylation and rebinding to nuclear structures of pRB takes more than 4 h. Within this time span, cells in G2 complete mitosis and divide. The slow dephosphorylation of pRB indicates that pRB is neither associated with the instantaneous inhibition of DNA synthesis nor is it the cause of the oxygen-dependent restriction point located in late-G1. The observed dephosphorylation of pRB is not dependent on functional p53, suggesting that at least one of the mechanisms responsible for the dephosphorylation is due to hypoxic activation of a pRB-specific phosphatase in the absence of p53-dependent inhibition of pRB kinase activity. However, it cannot be ruled out the participation of pRB kinase inhibitors independent of p53 activation. Cells arrested in G1 during prolonged hypoxia resumed cell-cycle progression within 2-->24 h after reoxygenation, while cells arrested in S were unable to reenter cell-cycle progression after reoxygenation. The hypoxia-induced dephosphorylation of pRB was only partly reversible by reoxygenation. Reentry into the cell cycle induced by reoxygenation occurred concomitant with unbinding (hyperphosphorylation) of pRB. Thus, rephosphorylation of pRB seem to be the rate-limiting step for reentry into the cell cycle after reoxygenation. Although pRB seems to play a major role in suppression of cell growth under and following hypoxic stress, other factors seem to be responsible for the immediate hypoxia-induced arrest in late-G1 and S phases.

Breast Neoplasms↗

PubMed1996

TNF-alpha bidirectionally modulates the viability of primitive murine hematopoietic progenitor cells in vitro.

It is well established that TNF-alpha can induce apoptosis in many normal and transformed cell types. The effects of TNF-alpha on cytokine-induced proliferation and differentiation of normal hematopoietic progenitors have been characterized extensively, whereas little is known about how TNF-alpha can affect their viability. The present studies suggest, based on experiments using delayed addition of growth-promoting cytokines as well direct viability assays, that TNF-alpha bidirectionally affects the survival of individually cultured primitive Lin- Sca-1+ hematopoietic progenitors, in that stem cell factor (SCF)-, granulocyte-CSF-, IL-6-, and IL-11-induced survival is potently counteracted by TNF-alpha (42-86%), whereas TNF-alpha synergistically enhances IL-1alpha-induced survival up to threefold. The bidirectional effects of TNF-alpha on hematopoietic growth factor-induced survival of hematopoietic progenitors were reflected in that TNF-alpha enhanced apoptosis of Lin- Sca-1+ cells when combined with SCF, whereas TNF-alpha synergistically suppressed apoptosis in response to IL-1alpha.

Animals↗