Counteraction of pRb-dependent protection after extreme hypoxia by elevated ribonucleotide reductase.

We have studied hypoxia-induced cell cycle arrest in human cells where the retinoblastoma tumour suppressor protein (pRb) is either functional (T-47D and T-47DHU-res cells) or abrogated by expression of the HPV18 E7 oncoprotein (NHIK 3025 cells). We have previously found that pRb is dephosphorylated and rebound in the nucleus in T-47D cells arrested in S-phase during hypoxia and that this binding is protracted even following re-oxygenation. In the present study, however, we show that the long-lasting arrest following re-oxygenation induced by pRb-binding in the cell nuclei may be overruled by an elevated level of ribonucleotide reductase (RNR). This seems to create a forced DNA-synthesis, uncoordinated with cell division, which induces endoreduplication of the DNA. The data indicate that the cells initiating endoreduplication continue DNA-synthesis until all DNA is replicated once and then may start cycling and cell division with a doubled DNA-content. Corresponding data on the pRb-incompetent NHIK 3025-cells show similar endoreduplication in these. Thus, the data indicate that endoreduplication of DNA following re-oxygenation may come, either as a result of hypoxic arrest of DNA-synthesis when pRb-function is absent in the cells, or if it is overruled by increased RNR. The present study further shows that pRb not only protects the culture by arresting most of the cells that are exposed to extreme hypoxia in S-phase, but also increases cell survival by means of increased clonogenic ability of these cells. Interestingly, however, cells having an elevated level of RNR have equally high survival as wild-type cells following 20 h extreme hypoxia. If RNR-overruling of pRb-mediated arrest following re-oxygenation results in an unstable genome, this may therefore represent a danger of oncogenic selection as the protective effect of pRb on cell survival seems to be maintained.

Lack of inverse dose-rate effect and binding of the retinoblastoma gene product in the nucleus of human cancer T-47D cells arrested in G2 by ionizing radiation.

PURPOSE: To investigate the radiosensitivity of human breast cancer cells, T-47D, irradiated with low dose-rates and to study activation of the retinoblastoma gene product in the G1 and G2 phases during irradiation. MATERIALS AND METHODS: Cells were irradiated with (60)Co gamma-rays with dose-rates of 0.37 and 0.94 Gy h(-1). Cell survival was measured as the ability of cells to form colonies. Cells were extracted, fixed and stained for simultaneous measurements of nuclear-bound pRB content and DNA content. Cell nuclei were stained with monoclonal antibody PMG3-245 and Hoechst 33258 was used for additional staining of DNA. Two-parametric flow cytometry measurements of pRB and DNA content were performed using a FACSTAR(PLUS) flow cytometer. RESULTS: It was observed that irradiated cells were arrested in G2. No increase in radiation sensitivity was observed when the cells accumulated in G2. Irradiation of cells at both 0.37 and 0.94 Gy h(-1) resulted in exponential dose-survival curves with nearly equal alpha values, i.e. the same radiosensitivity. However, the retinoblastoma gene product was bound in the nucleus, i.e. hypophosphorylated, in about 15% of the cells arrested in G2. CONCLUSIONS: T47-D cells accumulate in G2 during low dose irradiation, but no inverse dose-rate effect, i.e. a more efficient inactivation of cells at lower than at higher dose-rates, was observed. A population of arrested G2 cells has pRB protein bound in the nucleus, and pRB therefore could play a role in protecting cells against radiation-induced cell death in G2.

Hypoxia-induced irreversible S-phase arrest involves down-regulation of cyclin A.

We have studied hypoxia-induced cell cycle arrest in human cells where the retinoblastoma tumour suppressor protein (pRB) is either functional (T-47D cells) or abrogated by expression of the HPV18 E7 oncoprotein (NHIK 3025 cells). All cells in S phase are immediately arrested upon exposure to extreme hypoxia. During an 18-h extreme hypoxia regime, the cyclin A protein level is down-regulated in cells of both types when in S-phase, and, as we have previously shown, pRB re-binds in the nuclei of all T-47D cells (Amellem et al. 1996). Hence, pRB is not necessary for the down-regulation of cyclin A during hypoxia. However, our findings indicate that re-oxygenation cannot release pRB from its nuclear binding following this prolonged exposure. The result is permanent S-phase arrest even after re-oxygenation, and this is correlated with a complete and permanent down-regulation of cyclin A in the pRB functional T-47D cells. In contrast, both cell cycle arrest and cyclin A down-regulation in S phase are reversed upon re-oxygenation in non-pRB-functional NHIK 3025 cells after prolonged exposure to extreme hypoxia. Our results indicate that pRB is involved in permanent S-phase arrest and down-regulation of cyclin A after extreme hypoxia.

Cell cycle progression and radiation survival following prolonged hypoxia and re-oxygenation.

PURPOSE: To investigate cell cycle progression and radiation survival following prolonged hypoxia and re-oxygenation. MATERIALS AND METHODS: NHIK 3025 human cervical carcinoma cells were exposed to extremely hypoxic conditions (<4ppm O2) for 20 h and then re-oxygenated. The subsequent cell cycle progression was monitored by analysing cell cycle distribution at different time-points after re-oxygenation using two-dimensional flowcytometry. The clonogenic survival after a 3.6 Gy X-ray dose was also measured at each of these time-points. The measured radiation survival was compared with theoretical predictions based on cell cycle distribution and the radiation age response of the cells. RESULTS: Following re-oxygenation the cells resumed cell cycle progression, completed S-phase, and then accumulated in G2. Non-clonogenic cells remained permanently arrested in G2, while the remainder of the cells completed mitosis after a few hours delay. The radiation survival of the hypoxia-pretreated cell population remained lower than for an exponentially growing control population for the investigated 50h of re-oxygenation. However, following 7 h of re-oxygenation, the radiation survival of the hypoxia-treated cell population correlated well with theoretically predicted values based on cell cycle distribution and radiation age response. CONCLUSIONS: The work demonstrates that prolonged hypoxia followed by re-oxygenation results in a G2 delay similar to that observed after DNA damage. Furthermore, chronic hypoxia results in decreased radiation survival for at least 50h following the reintroduction of oxygen. The hypoxia-induced radiosensitization following 7 h of re-oxygenation could in large part be explained by the synchronous cell cycle progression that occurred.

Survival of synchronized human NHIK 3025 cells irradiated aerobically following a prolonged treatment with extremely hypoxic conditions.

PURPOSE: To investigate whether radiation survival of cells irradiated aerobically in the oxygen-sensitive restriction point in late G1 is dependent on where in the cell cycle the cells first were rendered hypoxic. MATERIALS AND METHODS: Human cervix carcinoma, NHIK 3025 cells, were synchronized and rendered hypoxic while in early-, mid- or late G1 or in early G2. Cell-cycle progression during the treatment was monitored by flow cytometry, and cell survival following either hypoxia alone or hypoxia with subsequent reoxygenation and irradiation was measured by the ability of the cells to form macroscopic colonies. RESULTS: During prolonged hypoxia, all surviving cells accumulated in an oxygen-sensitive restriction point in late G1. Cells rendered hypoxic in G2 initiated DNA synthesis following reoxygenation and irradiation several hours later than cells rendered hypoxic in G1. Radiation survival of cells accumulated in the oxygen-sensitive restriction point was independent of where in the cell cycle the cells first were rendered hypoxic. The hypoxia-treated cells had lower radiation survival probability than untreated cells in late G1. CONCLUSIONS: Although cells accumulated in the oxygen-sensitive restriction point from different parts of the cell cycle are not biologically identical, they are radiobiologically similar. The radiosensitizing effect of prolonged hypoxia was not merely due to cell-cycle redistribution.

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.

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.

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.

Regulation of cell proliferation under extreme and moderate hypoxia: the role of pyrimidine (deoxy)nucleotides.

In the present study we have used flow cytometric DNA measurements on synchronised human NHIK 3025 cells to measure cell cycle progression under various conditions of reduced oxygenation. Our data indicate that addition of 0.1 mM deoxycytidine or uridine has no effect on the oxygen-dependent arrest in late G1 or on the inhibition of cell proliferation through S-phase under extremely hypoxic conditions. Following reoxygenation of cells exposed to extremely hypoxic conditions in G2 initiation of DNA synthesis in the subsequent cell cycle is delayed by several hours. This G2-induced delay is completely abolished for approximately 60% of the cell population by addition of deoxycytidine to hypoxic G2 cells. This finding supports our previous proposal that important steps in the preparation for DNA synthesis occur during G2 of the previous cell cycle, and it indicates that this preparation is connected to the de novo synthesis of pyrimidine deoxynucleotide precursors. The results show that cells are able to enter S-phase in the presence of 100-1,300 p.p.m. (0.01-0.13%) oxygen (here denoted 'moderate hypoxia'), but they are not able to complete DNA synthesis under such conditions. However, the cell cycle inhibition induced under moderate hypoxia is partially reversed in the presence of exogenously added deoxycytidine and uridine, while no such reversal is seen in the presence of purine deoxynucleosides (deoxyadenosine and deoxyguanosine). Thus, both deoxycytidine and uridine could replace reoxygenation under these conditions. These results indicate that the reduction of CDP to dCTP by ribonucleotide reductase, an enzyme which requires oxygen as an essential factor for the formation of tyrosyl radicals for its catalytic activity, does not seem to be the limiting step responsible for the reduced dCTP pool observed under moderate hypoxia. We conclude that the oxygen-dependent catalytic activity of the M2 subunit of ribonucleotide reductase is still intact and functional in NHIK 3025 cells even at oxygen concentration as low as 100 p.p.m. Therefore the cell cycle inhibition observed is probably due to inhibition of the respiratory chain-dependent UMP synthesis at the stage of dihydroorotate dehydrogenase.

Hypoxia-associated proteins in human cells cultivated in vitro: lack of association with hypoxia-induced cell cycle regulation.

The synthesis of proteins expressed in human NHIK 3025 cells following exposure to extremely hypoxic conditions (< 4 ppm O2) has been studied. Populations of cells, either in exponential growth or synchronized by the method of detaching mitotic cells, were exposed to extremely hypoxic conditions for up to 20 h. The rate of total protein synthesis was measured at various time points after reoxygenation, and it appeared to be relatively constant and similar to the control level. The protein expression in cells was studied by pulse labelling for 1 h with [35S]-methionine, and subsequently visualized by SDS-PAGE and autoradiography. Six proteins appeared to have a changed expression after exposure to extreme hypoxia as compared to control cells; four of them (45, 80, 100 and 150 kD) showed increased, while two (46 and 90 kD) showed decreased expression. The response of these proteins to extreme hypoxia seems to be relatively slow, i.e. with half-times of several hours. Since extreme hypoxia influences cell cycle progression by instantaneous blockage at the G1/S border as well as halting DNA synthesis in S cells, these proteins can hardly cause these effects. Neither is the altered expression of these proteins due to the accumulation of G1 cells caused by hypoxia.

Cell cycle progression in human cells following re-oxygenation after extreme hypoxia: consequences concerning initiation of DNA synthesis.

The initiation of DNA synthesis and further cell cycle progression in cells during and following exposure to extremely hypoxic conditions in either G1 or G2 + M has been studied in human NHIK 3025 cells. Populations of cells, synchronized by mitotic selection, were rendered extremely hypoxic (< 4 p.p.m. O2) for up to 24 h. Cell cycle progression was studied from flow cytometric DNA recordings. No accumulation of DNA was found to take place during extreme hypoxia. Cells initially in G1 at the onset of treatment did not enter S during up to 24 h exposure to extreme hypoxia, but started DNA synthesis in a highly synchronous manner within 1.5 to 2.25 h after reoxygenation. The duration of S phase was only slightly affected (increased by approximately 10%) by the hypoxic treatment. This suggests that the DNA synthesizing machinery either remains intact during hypoxia or is rapidly restored after reoxygenation. Cells initially in G2 at the onset of hypoxia were able to complete mitosis, but further cell cycle progression was blocked in the subsequent G1. Following reoxygenation, these cells progressed into S phase, but the initiation of DNA synthesis was delayed for a period corresponding to at least the duration of normal G1 and did not appear in a synchronous manner. In fact, cell cycle variability was found to be increased rather than decreased as a result of exposure to hypoxia starting in G2. We interpret these findings as an indication that important steps in the preparation for initiation of DNA synthesis take place before mitosis. Furthermore, the change in cell cycle duration induced by hypoxia commencing in G1 is of a nature other than that induced by hypoxia commencing in other parts of the cell cycle.

Deoxyribonucleic acid cytometry and histological findings before and after 125iodine implantation of primary prostate cancer.

Deoxyribonucleic acid (DNA) flow cytometry and light microscopy were performed in pre-radiotherapy and post-radiotherapy biopsies obtained from the primary tumor in 31 patients with prostate cancer. Radiotherapy was applied by means of transperineal 125iodine (125I) implantation. Of the patients 21 had pretreatment biopsies and in 19 of these biopsies also were performed 1 and/or 1 1/2 years after the 125I implantation. Posttreatment biopsies were available for DNA flow cytometry in 12 additional patients without pretreatment DNA flow cytometry assessment. Of the 21 pretreatment biopsies 7 were diploid, 6 tetraploid and 8 aneuploid. All 31 posttreatment biopsies were either tetraploid (21) or aneuploid (10). All 6 pretreatment diploid tumors became tetraploid after radiotherapy. At 1 and/or 1 1/2 years after 125I implantation residual tumors were found in 28 of 31 prostatic glands. The high frequency of nondiploid DNA stemlines 1 or more years after 125I implantation and the high rate of residual tumor leave some doubt about the radiocurability of prostate cancer by the chosen radiotherapy technique.

The prognostic significance of deoxyribonucleic acid flow cytometry in muscle invasive bladder carcinoma treated with preoperative irradiation and cystectomy.

Deoxyribonucleic acid (DNA) flow cytometry measurements were performed in nuclear suspensions obtained from paraffin-embedded biopsies from 83 patients with stages T2, T3 and T4a bladder carcinoma. All patients were treated with preoperative radiotherapy and cystectomy from 1976 through 1985. Of the tumors 13 (16%) were diploid, 18 (22%) tetraploid and 52 (63%) aneuploid. A total of 19 tumors (23%) had 2 or 3 stemlines in addition to the diploid cells. Post-radiotherapy stage reduction (absence of muscle infiltration in the cystectomy specimen) occurred more often in tumors with only 1 nondiploid stemline than in diploid tumors or nondiploid tumors with multiple stemlines. The 5-year survival rate was significantly poorer for patients with a diploid (33%) than for those with a nondiploid (66%) tumor (p = 0.05), although this was only marginally retained in a multivariate analysis (p = 0.11). The clinical significance of DNA ploidy in muscle infiltrating bladder cancer seems not to be as evident as has been shown for superficial bladder tumors but it may be of value in selecting patients for preoperative radiotherapy.

DNA ploidy in primary testicular cancer.

The DNA stemline ploidy was measured by flow cytometry (FCM) in 129 samples from paraffin-embedded primary testicular tumours (61 seminomas, 68 non-seminomas). Only one DNA stemline was found in 38 seminomas and 44 non-seminomas. Two seminomas and one non-seminoma were DNA diploid, the other tumours being non-diploid. Twenty-three seminomas and 24 non-seminomas displayed two or three DNA stemlines. The median minimal DNA index (DI) of all seminomas was significantly higher than that of all non-seminomas (1.58 vs 1.43; P: 0.008). Three seminomas removed from two monozygotic twins within 1 week had DIs of 1.66, 1.56 and 1.59. In this limited series there was no association between DNA ploidy of the primary tumour and the metastatic status for either seminomas or non-seminomas. The results support the pathogenetic model stating that at least some (if not all) non-seminomas develop from a seminoma by additional chromosomal aberration. The clinical relevance of DNA stemline ploidy has to be further evaluated in larger series.

The role of protein accumulation on the kinetics of entry into S phase following extreme hypoxia.

Protein accumulation was measured in human NHIK 3025 cells, synchronized by mitotic selection, during and following treatment with extreme hypoxia (less than 4 ppm O2). Comparison was made between cells treated while in different stages of the G1 phase. The total protein content was measured in individual cells and related to cell cycle phase by coincident recording of DNA and protein using two-parametric flow cytometry. After a 24 h treatment with extreme hypoxia, all cells, irrespective of where in G1 they were located at the start of treatment, were reversibly arrested in a pre NDA synthetic stage. During this treatment there was a slight reduction (approximately 2%) in the average amount of protein per cell. The arrested cells were found to display a heterogeneous protein content after treatment, but cells rendered hypoxic while in early-G1, had clearly lost more protein during hypoxia than cells rendered hypoxic while in late-G1. After reoxygeneration, the cells initiated DNA synthesis after first having restored the amount of protein which is normal for these cells at the stage of initiation. Thus, in the first hours following reoxygenation, cells rendered hypoxic while in early-G1 accumulated protein faster than those rendered hypoxic while in late-G1 (the respective rates being 12.3 versus 5.2%/h). We conclude that this protein accumulation may be of importance in restoring the pool of protein required for the initiation of DNA synthesis and that the differences in rate reflect the amount of protein lost during hypoxia.

DNA ploidy in the primary tumor from patients with nonseminomatous testicular germ cell tumors clinical stage I.

The DNA stemline ploidy was assessed in paraffin-embedded, formalin-fixed primary tumor tissue from 68 patients with nonseminomatous germ cell cancer (NSCGT) clinical Stage I (CS I). Forty-three patients had a single aneuploid (greater than 1C) DNA stemline, whereas 24 patients had multiple aneuploid stemlines. In one tumor there was no evidence of an abnormal DNA stemline. Most DNA stemlines had DNA indices around the 3c value. In 13 patients there was a good correlation between the DNA stemline values observed in the primary tumor and in the retroperitoneal lymph node metastases. No correlation was found between the DNA index and the histologic subclassification or the metastatic behavior. The size of the S-phase fraction did not appear to be predictive of subclinical metastases. In CS I patients with NSCGT determination of DNA stemline values does not yield information of predictive or prognostic significance but may contribute to the understanding of the pathogenesis of NSCGT.

Cell inactivation and cell cycle inhibition as induced by extreme hypoxia: the possible role of cell cycle arrest as a protection against hypoxia-induced lethal damage.

Cycling mammalian cells that are rendered extremely hypoxic (less than 4 ppm O2) tend to accumulate in a pre-DNA-synthesis stage. It is not clear whether or not this is the result of an active regulation by the cells. In the present study we have rendered cells, synchronized by mitotic selection, extremely hypoxic over a relatively long period of time (up to 48 h). We have recorded cell cycle progression during hypoxia as well as cell inactivation depending on where in the cell cycle the cells were located when the hypoxic treatment was started. Three main conclusions are drawn: 1 the cell cycle arrest in late-G1 is complete even during a long-lasting (24 h) hypoxic treatment: 2 while cells in early- and mid-S are completely arrested and quickly inactivated under hypoxic conditions, cells in late-S, G2 and mitosis are able to continue cell cycle progression and divide; 3 whether the cells are located in G2, mitosis or early-G1 at the onset of hypoxia, they were able to survive relatively long-lasting hypoxic treatment. The present results are in favour of the view that the pre-DNA-synthetic arrest induced by extreme hypoxia may function to rescue the cells from severely damaging effects that would appear if the cells were able to initiate DNA synthesis.

DNA flow cytometry in sperm cells from testicular cancer patients. Impact of different treatment modalities on spermatogenesis.

In 77 patients with testicular cancer, sperm cell density was evaluated before and after treatment together with the following parameters as recorded by DNA flow cytometry: the percentage of condensed and non-condensed haploid sperm cells, and the number of non-haploid cells. The patients received either abdominal radiotherapy, cisplatin-based combination chemotherapy (+/- surgery) or did not undergo antiproliferative treatment at all. Ejaculates with a low sperm cell density had high numbers of non-condensed haploid and non-haploid cells, whereas the percentage of condensed haploid cells was decreased. However, even 'azoospermic' semen samples (by light microscopy) contained haploid cells indicating ongoing sperm cell production. One year after radiotherapy there was a significant decrease in the sperm cell density and the number of condensed haploid cells. Chemotherapy led to a significant reduction of sperm cell density evaluated 1 year after treatment. Three years after the diagnosis of testicular cancer, sperm cell production had recovered in most patients. In patients who did not have radiotherapy or chemotherapy the median density of sperm cells 3 years after treatment significantly exceeded the corresponding figure from the pretreatment situation. A low pretreatment percentage of condensed haploid cells (less than or equal to 90%) was correlated with the lack of 1-year recovery of sperm cell production. It is too early to state whether longitudinal flow cytometric data from ejaculates in testicular cancer patients yield clinically valuable information, in addition to that obtained by light microscopy. From this preliminary observation it seems that DNA flow cytometry in ejaculates from testicular cancer patients may give valuable clinical information about sperm cell production following treatment.