Pericellular oxygen monitoring with integrated sensor chips for reproducible cell culture experiments.

OBJECTIVES: Here we present an application, in two tumour cell lines, based on the Sensing Cell Culture Flask system as a cell culture monitoring tool for pericellular oxygen sensing. MATERIALS AND METHODS: T-47D (human breast cancer) and T98G (human brain cancer) cells were cultured either in atmospheric air or in a glove-box set at 4% oxygen, in both cases with 5% CO2 in the gas phase. Pericellular oxygen tension was measured with the help of an integrated sensor chip comprising oxygen sensor arrays. RESULTS: Obtained results illustrate variation of pericellular oxygen tension in attached cells covered by stagnant medium. Independent of incubation conditions, low pericellular oxygen concentration levels, usually associated with hypoxia, were found in dense cell cultures. CONCLUSIONS: Respiration alone brought pericellular oxygen concentration down to levels which could activate hypoxia-sensing regulatory processes in cultures believed to be aerobic. Cells in culture believed to experience conditions of mild hypoxia may, in reality, experience severe hypoxia. This would lead to incorrect assumptions and suggests that pericellular oxygen concentration readings are of great importance to obtain reproducible results when dealing with hypoxic and normoxic (aerobic) incubation conditions. The Sensing Cell Culture Flask system allows continuous monitoring of pericellular oxygen concentration with outstanding long-term stability and no need for recalibration during cell culture experiments. The sensor is integrated into the flask bottom, thus in direct contact with attached cells. No additional equipment needs to be inserted into the flask during culturing. Transparency of the electrochemical sensor chip allows optical inspection of cells attached on top of the sensor.

Oxygen consumption in T-47D cells immobilized in alginate.

OBJECTIVES: Encapsulation or entrapment of cells is increasingly being used in a wide variety of scientific studies for tissue engineering and development of novel medical devices. The effect on cell metabolism of such systems is, in general, not well characterized. In this work, a simple system for monitoring respiration of cells embedded in 3-D alginate cultures was characterized. MATERIALS AND METHODS: T-47D cells were cultured in alginate gels. Oxygen concentration curves were recorded within cell-gel constructs using two different sensor systems, and cell viability and metabolic state were characterized using confocal microscopy and commercially available stains. RESULTS: At sufficient depth within constructs, recorded oxygen concentration curves were not significantly influenced by influx of oxygen through cell-gel layers and oxygen consumption rate could be calculated simply by dividing oxygen loss in the system per time, by the number of cells. This conclusion was supported by a 3-D numeric simulation. For the T-47D cells, the oxygen consumption rate was found to be 61 ± 6 fmol/cell/h, 3-4 times less than has previously been found for these cells, when grown exponentially in monolayer culture. CONCLUSIONS: The experimental set-up presented here may be varied in multiple ways by changing the cell-gel construct 3-D microenvironment, easily allowing investigation of a variety of factors on cell respiration.

Response of chronic hypoxic cells to low dose-rate irradiation.

PURPOSE: Compare the sensitivity of human cells in vitro to low dose-rate irradiation in air and in moderate hypoxia (4% O2). MATERIALS AND METHODS: Continuous low dose-rate beta-irradiation at a dose rate of 0.015 or 0.062 Gy/h was given to human T-47D breast cancer cells by incorporation of [3H] -labelled valine into cellular protein. Acute irradiation at a dose rate of 0.4 Gy/min was performed using [137Cs]gamma-irradiation. Cells were cultivated in an atmosphere with 4% O2 using an INVIVO2 hypoxia cabinet. RESULTS: When grown in ambient air with continuous irradiation, T-47D cells were able to continue growth for at least 23 weeks at a dose-rate of 0.015 Gy/h with a surviving fraction stabilized at around 60%. When the dose rate was increased to 0.062 Gy/h the cell culture died out after about 23 days (corresponding to about 22 Gy). When grown in an atmosphere with 4% O2 we surprisingly found that the continuously irradiated T-47D cells (0.015 Gy/h) were severely inhibited in their growth, and cell death became extensive after about 3 weeks while un-irradiated cells continued growth seemingly unaffected by this low oxygenation. Peri cellular oxygenation varied between 4% and below 0.1% over an ordinary passage due to diffusion-limitations through the 2 mm deep medium. Online O2-recordings over a whole passage showed that oxygen was more depleted in the irradiated compared to the un-irradiated cultures indicating increased respiration during irradiation. While cells growing attached to the bottom were inhibited and inactivated during irradiation it was found that cells attached high up in the neck region, i.e., having only a shallow layer of medium above them, survived and formed colonies. When cells cultivated in 4% O2 for 7 weeks were irradiated with acute doses of 137Cs gamma-rays, the radiosensitivity was the same as for cells cultivated in ambient air. CONCLUSION: Continuous irradiation with 0.015 Gy/h for several weeks results in a stronger inhibition for T-47D cells grown in an atmosphere with 4% as compared to 20% O2. The data indicate that this may be due to increased oxygen consumption resulting in more severe hypoxia in [3H]-incorporating compared to control (un-irradiated) cells.

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.

Histologic subtype has minor importance for overall survival in patients with adenocarcinoma of the uterine cervix: a population-based study of prognostic factors in 505 patients with nonsquamous cell carcinomas of the cervix.

BACKGROUND: The incidence of adenocarcinoma of the uterine cervix is increasing. For better prognostic information, the authors studied all nonsquamous cell carcinomas (non-SCCs) in the Norwegian population over a total of 15 years. METHODS: All non-SCCs from three 5-year periods (1966-1970, 1976-1980, and 1986-1990) were reviewed and classified according to the World Health Organization classification system, and histopathologic and clinical parameters were registered. Tissue blocks were available from all patients. RESULTS: Of 505 patients, 417 had tumors classified as adenocarcinoma, and 88 had tumors classified as other non-SCC. The mean ages were 53 years and 52 years for patients with adenocarcinoma and non-SCC, respectively. Sixty-two percent of the staged patients had clinical Stage I disease according to the classification system of the International Federation of Gynecology and Obstetrics (FIGO). In univariate analyses, histology, architectural and nuclear grade, extension to the vagina or corpus uteri, tumor length (> 20 mm) or tumor volume (> 3000 mm(3)), infiltration depth (in thirds of the cervical wall), thickness of the remaining wall (< 3 mm), vascular invasion, lymph node metastases, treatment, and patient age were significant variables in patients with FIGO Stage I disease. Variables with no significance in patients with Stage I disease were number of mitoses, state of resection margins, infiltration to ectocervix, tumor thickness, lymphoid reaction, earlier or concomitant cervical intraepithelial neoplasia, stump carcinoma, DNA ploidy or DNA index, or time period. Multivariate analyses of patients with FIGO Stage I disease identified small cell carcinoma, corpus infiltration, vascular invasion, and positive lymph nodes as independent prognostic factors. CONCLUSIONS: Small cell carcinoma was the only histologic subgroup of independent importance for prognosis in patients with non-SCC of the uterine cervix. No significant difference between major subtypes of adenocarcinoma favored a simplified classification. Extension to the corpus in patients with early-stage disease was of independent significance and should be acknowledged in planning treatment.

pH-dependent spectral properties of HpIX, TPPS2a, mTHPP and mTHPC.

Lower extracellular pH in tumors as compared to normal tissues has been proposed to be a factor contributing to the tumor selective uptake of several photosensitizers. Therefore, the pH dependence of absorption and fluorescence spectral properties of four different drugs relevant for photodynamic therapy (hematoporphyrin IX [HpIX], disulfonated meso-tetraphenylporphine [TPPS2a], meso-tetra(3-hydroxyphenyl)porphine [mTHPP] and meso-tetra(3-hydroxyphenyl)chlorin [mTHPC]) has been examined. Spectral analysis of the dyes dissolved in phosphate buffered saline (PBS) indicates pH-dependent modification in the physiologically important region (6.0-8.0) only in the case of HpIX. This modification is probably related to the protonation of carboxylic groups. Spectral changes of HpIX in PBS observed at acidic pH values < 5, as well as those of the rest of the drugs (inflection points of titration curves occurred at about 5.1, 3.8 and 2.4 for TPPS2a, mTHPP and mTHPC, respectively), are likely to be due to the protonation of imino nitrogens. The tumor localizing properties of mTHPP and mTHPC reported in the literature appear to be due to factors other than pH-dependent changes in the lipophilicity of the drugs.

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.

Measurement of dose rate at the interface of cell culture medium and glass dishes by means of ESR dosimetry using thin films of alanine.

Previous studies on human cervical cancer cells (NHIK 3025) have indicated that the cells, when X-irradiated in suspension, appeared to be more radiosensitive than when they were irradiated attached to glass dishes. However, this result depends on dosimetry, which is difficult in the situation where cells are attached to glass dishes due to backscattering electrons at the glass-liquid interface. Recently developed dosimetry that is based on detection of radiation-induced stable radicals in alanine and uses ESR spectroscopy offers a possibility for more relevant dosimetry at the glass-liquid interface than the previous estimates of doses based on Fricke dosimetry. Thin alanine films (>/=10 microm) were used to measure dose at the interface by irradiating the films while they were placed tightly against the bottom of dishes and covered with 1 mm of wax simulating the medium above cells. Fricke dosimetry was also performed, with different depths of Fricke solution in the dishes, to elucidate the contribution to the dose delivered by backscattering electrons at the glass-liquid interface. A dose rate of 1.9 Gy/min was measured with a thin layer (0.2-0.3 mm) of Fricke solution in petri dishes made of glass. However, this estimate appears to be too high, due to a contribution to dose by short-ranged electrons generated when the X rays passed through a steel lid 4.5 cm above the dishes. Dosimetry using alanine films resulted in dose rates of 1.15 and 0.87 Gy/min at the interfaces of glass-liquid and plastic- liquid, respectively. Hence there is a significant contribution to dose from backscattering electrons on dishes made of glass. The reason for our previous observation of a difference in radiosensitivity between cells irradiated in suspension and cells irradiated attached to glass appears to be a lack of accurate dosimetry at the glass-liquid interface.

Spermatocytic seminoma as compared to classical seminoma: an immunohistochemical and DNA flow cytometric study.

UNLABELLED: Based on immunohistochemistry (IHC) and DNA ploidy, different paths of carcinogenesis have been suggested for spermatocytic seminoma (SS) and classical seminoma (CS). The present study extends current knowledge on the above parameters. METHOD: Seventeen SSs and twenty-two CSs were assessed by IHC for placental-like alkaline phosphatase (PLAP), c-kit, cytokeratin and adhesion carbohydrate molecyles. All SSs and 11 CSs were also analysed for DNA ploidy. RESULTS: All CSs, but none of the SSs, were positive for PLAP. C-kit positivity was found in 7 of 17 SSs and in all CSs. The other IHC parameters were similarly distributed among the evaluated SSs and CSs. Fourteen SSs were diploid or polyploid, and three were aneuploid. All CSs were aneuploid. CONCLUSION: The new observation of c-kit positivity in about 40% of SSs suggests that at least some of the SSs originate from primordial cells. The predominantly diploid or polyploid DNA pattern indicates that SSs follow a pathogenetic pathway which is most probably different from that of CSs.

p53 protein expression in squamous cell carcinoma of the vulva.

BACKGROUND: The objective of the study was to evaluate the pathogenetic and prognostic value of p53 protein expression in squamous cell carcinoma of the vulva. METHODS: The clinical data in charts of 167 patients with International Federation of Gynecology and Obstetrics (FIGO) Stages I-III primary tumors who were treated by surgery were reviewed. Samples from the primary tumor were immunostained for p53 protein. p53 overexpression was defined as immunoreactivity in > 5% of nuclei. RESULTS: p53 overexpression was observed in 92 tumors (55%). p53 overexpression did not correlate with age at diagnosis, FIGO stage, histologic grade, vessel invasion, tumor thickness, tumor greatest dimension, DNA ploidy, or inguinal lymph node metastasis. In the whole group a significantly reduced 5-year corrected survival was observed in patients with p53 overexpression compared with p53 negative patients (P = 0.04). In the different FIGO stages, disease-related survival was not influenced by p53 overexpression in 37 patients with Stage I disease (P = 0.60) or in 86 patients with Stage II disease (P = 0.96). In 44 patients with Stage III disease, p53 overexpression was significantly associated with poorer prognosis (P = 0.004). Independent prognostic factors for corrected survival in the entire group of 167 patients were: vascular invasion, groin metastasis, tumor greatest dimension, and p53 overexpression. In patients with FIGO Stage III disease p53 overexpression was not an independent prognostic factor. CONCLUSIONS: p53 protein overexpression appears to be involved in the pathogenesis of vulvar squamous cell carcinoma. p53 protein overexpression was significantly associated with disease-related survival. p53 prognostic impact was observed only in patients with advanced disease.

Cell adhesion force microscopy.

The adhesion forces of cervical carcinoma cells in tissue culture were measured by using the manipulation force microscope, a novel atomic force microscope. The forces were studied as a function of time and temperature for cells cultured on hydrophilic and hydrophobic polystyrene substrates with preadsorbed proteins. The cells attached faster and stronger at 37 degreesC than at 23 degreesC and better on hydrophilic than on hydrophobic substrates, even though proteins adsorb much better to the hydrophobic substrates. Because cell adhesion serves to control several stages in the cell cycle, we anticipate that the manipulation force microscope can help clarify some cell-adhesion related issues.

Inverse dose-rate effect due to pre-mitotic accumulation during continuous low dose-rate irradiation of cervix carcinoma cells.

PURPOSE: To investigate the radiation sensitivity of asynchronous and synchronized cancer cervix cells irradiated with low dose rates. MATERIALS AND METHODS: Cells were exposed to 60Co gamma-rays at dose rates ranging from 0.33 to 0.94 Gy/h. Synchronized cells were obtained by collecting detached mitotic cells after a shaking procedure. Cell survival was measured as the ability of cells to form colonies. Cell-cycle distributions were calculated by computer analysis of a DNA histogram recorded by flow cytometry. RESULTS: Irradiation of asynchronous cells at either 0.33 or 0.86 Gy/h resulted in exponential dose-survival curves with equal alpha-values, i.e. same radiation sensitivity, when dose-survival data for irradiation periods less than 20h were considered. However, the radiation sensitivity was higher by a factor of two when analysing dose-survival data for irradiation periods exceeding 20h. This increase in radiation sensitivity occurred when 80% of the cells accumulated in a pre-mitotic stage of the cell cycle. Irradiation of synchronized cell populations confirmed that these cells were a factor of two more sensitive to radiation in G2 than in G1. CONCLUSIONS: An inverse dose-rate effect, i.e. more efficient inactivation of cells at lower rather than at higher dose rates, was observed for radiation doses exceeding 7 Gy due to pre-mitotic accumulation of cells during low dose-rate irradiation.

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.

Synovial sarcoma. Evaluation of prognosis with emphasis on the study of DNA ploidy and proliferation (PCNA and Ki-67) markers.

Controversy still exists regarding the validity of parameters commonly used in the evaluation of prognosis of patients with synovial sarcoma (SS). Forty-nine cases of previously untreated primary SS (23 females and 26 males, ranging in age from 7 to 81, with 31 tumors located in the lower extremity, 8 at the upper extremity and 10 at the trunchus), without regional lymph-node or distant metastases were studied. We investigated the relationship between (flow and image) DNA cytometry, proliferation activity, clinicopathologic parameters, and relapse-free and overall survival of the patients. The prognostic value of gender, age, duration of symptoms, location, compartmentalization, size, adequacy of surgical margins, residual tumor, adjuvant therapy, histologic subtype, extent of necrosis, glandular differentiation, calcification, and extent of hemangiopericytic areas, mitotic rate, amount of mast cells, blood vessel invasion, histologic (UICC and NCI) grades, DNA ploidy, percentage of cells in S and S+G2 phases, PCNA and Ki-67 labeling indices (LI), and TNM (UICC) stage of the tumors, were evaluated by univariate and multivariate (Cox hazard model) analyses. Short duration of symptoms (<12 months), biphasic SS, scarcity of mast cells (<10/10 HPF), high mitotic rate (> or =10/10 HPF), high histologic grade (grade 3), high PCNA-LI (> or =20%), high Ki-67-LI (> or =10%), DNA aneuploidy, and advanced TNM stage (stage III) were features associated with significantly shorter relapse-free and overall 5-year survival rates in the univariate analyses. Scarcity of mast cells, high mitotic rate, or high PCNA-LI were significant predictors of poor survival, in addition to TNM stage in the multivariate analyses. The amount of mast cells was inversely correlated with mitotic rate and PCNA-LI. Scarcity of mast cells, high mitotic rate, or high PCNA-LI are factors associated with poor prognosis, in addition to advanced TNM stage in patients with localized SS.

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.