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.

Synergistic cell inactivation of human NHIK 3025 cells by cinnamaldehyde in combination with cis-diamminedichloroplatinum(II).

The cell-inactivating effect induced by cinnamaldehyde in combination with cis-diamminedichloroplatinum(II) (cis-DDP) on human NHIK 3025 cells in culture was investigated. Cell inactivation was measured as a loss in the ability of single cells to give rise to macroscopic colonies following drug treatment. Although 2 h treatment of asynchronous cells with 0.3 mM cinnamaldehyde alone induced little cell inactivation, the drug combination of 0.3 mM cinnamaldehyde and 10 microM cis-DDP resulted in synergistic cell inactivation. Cinnamaldehyde potentiated the cell-inactivating effect of cis-DDP by a dose-modifying factor of 1.8. Drug synergism was found to occur only when cinnamaldehyde and cis-DDP were given in simultaneous combination. Treatment of synchronized cells demonstrated that cinnamaldehyde potentiated the inactivating effect of cis-DDP in all phases of the cell cycle. Cinnamic acid and cinnamyl alcohol were found to have no synergistic or potentiating effect on cell survival following treatment of cells with cis-DDP, thus indicating the importance of an aldehyde moiety for drug synergism.

4,6-Benzylidene-D-glucose, a benzaldehyde derivative that inhibits protein synthesis but not mitosis of NHIK 3025 cells.

The effect of the benzaldehyde derivative 4,6-benzylidene-D-glucose (BG) on cell-cycle kinetics, protein synthesis, and cell survival of human NHIK 3025 cells has been investigated. The effects are compared with results reported previously for benzaldehyde, which was shown to inhibit protein synthesis as well as induce metaphase inhibition. BG exerted an effect on protein synthesis similar to that of benzaldehyde, but did not affect metaphase. Inhibition of protein synthesis was measured as a reduction in the rate of incorporation of [3H]valine and is thus a measure of the rate of total protein synthesis. The quantitative effect on the rate of protein synthesis was equal for the two drugs when compared on a concentration basis. Both drugs induced inhibition of cell-cycle progression during interphase, which is thought to be a secondary result of the protein synthesis inhibition. The concentration of BG and the hydrolysis product benzaldehyde were determined in cell culture medium by high-performance liquid chromatography. BG was not found to be hydrolyzed to free benzaldehyde when in contact with cells or in a cell sonicate. The results indicate that BG is not metabolized but acts in the form of BG.

Regulation of protein metabolism of human cells during and after acute hypoxia.

Protein synthesis and protein degradation were measured in human NHIK 3025 cells cultured in vitro during and after an acute treatment of extreme hypoxia (less than 4 ppm O2). Furthermore, total protein content per cell was recorded and related to cell cycle phase by coincident measurement of DNA and protein using two-parametric flow cytometry. During hypoxia protein synthesis was reduced and protein degradation was increased, resulting in no net accumulation of protein. From the flow cytometric recordings, the amount of protein per cell was found to be constant, or perhaps in some of the cells slightly reduced, after a 3-h period of extreme hypoxia. Three h after reaeration protein degradation had returned to normal while protein synthesis was slightly above normal. The flow cytometric recordings showed that after reaeration the protein accumulation was particularly high in the subpopulation of cells which accumulated at the G1-S border during hypoxia and entered S phase as a partly synchronized subpopulation after reaeration. Since we know from our earlier studies that these cells are more resistant to hypoxia than cells in S phase we conclude that this high protein accumulation may be important in restoring a pool of proteins which initiate DNA synthesis and perhaps other proteins of importance to cell growth.

Modifying effect of cinnamaldehyde and cinnamaldehyde derivatives on cell inactivation and cellular uptake of cis-diamminedichloroplatinum(II) in human NHIK 3025 cells.

The effect of cinnamaldehyde and various cinnamaldehyde derivatives on cell inactivation induced by cis-diamminedichloroplatinum(II) (cis-DDP) was investigated using human NHIK 3025 cells in culture. Cell inactivation was measured as a loss in the ability of single cells to give rise to macroscopic colonies following drug treatment. It was found that cinnamaldehyde and alpha-chlorocinnamaldehyde potentiated the cell-inactivating effect when used simultaneously with cis-DDP without increasing the amount of cell-associated platinum. In contrast, a protective effect with respect to cell inactivation was found when cells were treated with cis-DDP in combination with hydrocinnamaldehyde or alpha-methylcinnamaldehyde. At higher concentrations (greater than 1 mM) all cinnamaldehyde derivatives reduced cellular uptake of cis-DDP. Therefore, while protection from cis-DDP-induced cell inactivation involves reduced platinum uptake, potentiation by cinnamaldehyde and cinnamaldehyde derivatives does not seem to be due to an increase in intracellular platinum. We propose that cinnamaldehyde may compete with cis-DDP in nucleophilic addition reactions involving intracellular sulfhydryls.

Isolation of carcinogen-induced diploid rat hepatocytes by centrifugal elutriation.

The majority of hepatocytes isolated from rats treated with carcinogens (diethylnitrosamine plus 2-acetylaminofluorene) were found to be diploid, whereas most of the hepatocytes from normal rats are tetraploid. The carcinogen-induced diploid hepatocytes were only one-half the size (protein content) of the tetraploid hepatocytes, and could therefore be separated from the latter by centrifugal elutriation. The elutriation technique thus makes it possible to isolate a relatively pure fraction of carcinogen-induced cells. The diploid cells had the same liver-specific enzymatic and functional properties as the tetraploid cells and were thus undoubtedly of hepatocytic origin.

Inhibitory and cytotoxic effects of Oncovin (Vincristine sulfate) on cells of human line NHIK 3025.

The inhibitory effect of Oncovin (vincristine sulfate) on cell division was studied in human cell line NHIK 3025. Oncovin arrested the cells in metaphase at concentrations as low as 10(-3) mug/ml. At 8 X 10(-3) mug/ml and higher concentrations, the arrest was complete after 6 hr of treatment. The arrest was irreversible after exposure for 6 hr to 16 X 10(-3) mug Oncovin per ml. The X-ray radiosensitivity of aerobic cells of the same line pretreated with 16 X 10(-3) mug Oncovin per ml for 6 hr (Oncovin removed before irradiation) was found to be about equal to that of untreated cells. Even when present during irradiation, Oncovin did not exert any modifying effect on the radiosensitivity of either aerobic or extremely hypoxic cells.

Interaction of 1-propargyl-5-chloropyrimidin-2-one (a metahalone) with rat brain tubulin.

The mitotic inhibitor 1-propargyl-5-chloropyrimidin-2-one (a metahalone) was found to bind to DEAE-cellulose purified rat brain tubulin. A decrease in the fluorescence of 1-propargyl-5-chloropyrimidin-2-one was seen when the drug was incubated in the presence of increasing tubulin concentrations. The decrease in metahalone fluorescence was not affected by the addition of GTP, indicating drug interaction at other portions of the tubulin molecule than the nucleotide binding sites. Scatchard plot analysis following incubation of tubulin with 1-propargyl-5-chloro-[2-14C]pyrimidin-2-one revealed that 1 mol of metahalone bound to 1 mol of tubulin dimer with a measured association constant of 8.0 X 10(3) M-1. Double reciprocal plots of vincristine and colchicine binding to tubulin in the presence of 1-propargyl-5-chloropyrimidin-2-one showed that the metahalone competitively inhibited colchicine binding to tubulin but had no influence on vincristine binding. This conclusion was supported by gel filtration chromatography where an increase in unbound colchicine was measured when 1-propargyl-5-chloropyrimidin-2-one was present in an incubation mixture containing colchicine and tubulin. In the presence of 5 mM 1-propargyl-5-chloropyrimidin-2-one, tubulin self-aggregated into crystalline structures. The binding of 1-propargyl-5-chloropyrimidin-2-one to tubulin at or near the colchicine binding site may be responsible for the metaphase arresting characteristics of this drug.

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.

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.

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.

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.

T2/T3 bladder carcinomas treated with definitive radiotherapy with emphasis on flow cytometric DNA ploidy values.

From 1972 through 1982, 124 patients with muscle invasive T2/T3 bladder carcinoma without evidence of distant metastases were treated with definitive radiotherapy (60 Gy in 6 weeks) to the pelvis at The Norwegian Radium Hospital. Sections from paraffin embedded biopsies taken prior to the start of treatment were prepared for DNA flow cytometry, and 121 histograms were considered interpretable for DNA ploidy values. Significantly improved survival was seen in patients with the following characteristic, univariate analysis: T2 tumor, macroscopically complete removal of the tumor prior to radiotherapy, clinically complete response to radiotherapy, normal serum creatinine, absence of hydronephrosis, tetraploid tumor, WHO performance status 0 or 1, age below 65 years. In a multivariate analysis the following pretreatment variables turned out with prognostic power in this order: normal serum creatinine, T2 tumor, tetraploidy, and absence of small vessel invasion. When including response to therapy, the following factors were significantly predictive of a beneficial survival: clinically complete response after radiotherapy, normal creatinine, T2 tumor and tetraploidy. Tetraploidy was associated with response to radiotherapy when compared to diploid tumors (p = 0.07). This relationship alone did not explain the better survival of patients with tetraploid tumors, and other factors concerned with "tumor aggressivity" may be additional explanatory parameters. DNA ploidy values provide valuable information in the pretreatment situation concerning the prognosis for patients with T2/T3 bladder carcinoma.

Protection from cis-dichlorodiammineplatinum-induced cell inactivation by aldehydes involves cell membrane amino groups.

The effect of cis-dichlorodiammineplatinum (cis-DDP) alone or in combination with the vitamin B6 analogs pyridoxal, pyridoxal 5'-phosphate and pyridoxine was tested on human NHIK 3025 cells. Cell inactivation was measured as loss of colony-forming ability following 2 h drug treatment. The cell inactivating effect of cis-DDP was significantly reduced when the aldehyde analogs pyridoxal or pyridoxal 5'-phosphate were simultaneously present with cis-DDP. The alcohol analog pyridoxine, however, did not affect cis-DDP-induced cell inactivation to any degree. Pyridoxine, pyridoxal or pyridoxal 5'-phosphate treatment alone had no effect on cell survival. Both pyridoxal and pyridoxal 5'-phosphate form Schiff bases with protein amino groups, but pyridoxal 5'-phosphate is cell membrane-impermeable. Therefore protection from cis-DDP-induced cell inactivation by aldehydes appears to involve cell membrane amino groups.

Modulation of cis-dichlorodiammineplatinum(II)-induced cytotoxicity by benzaldehyde derivatives.

The cell inactivating effect of cis-dichlorodiammineplatinum(II) in combination with 2-substituted benzaldehyde derivatives was tested using cultured human NHIK 3025 cells. Simultaneous drug combination with benzaldehyde protected cells from cis-DDP-induced cell inactivation in a dose-dependent manner. Substitution with electron-releasing groups, i.e. -CH3 and -OH, adjacent to the aldehyde moiety increased the protective effect relative to benzaldehyde. Electron-withdrawing, or bulky, substituents reduced the protective effect. Protection from cell inactivation by cis-DDP coincided with an aldehyde-mediated reduction in platinum accumulation.

DNA flow cytometry in human testicular cancer.

DNA flow cytometry revealed aneuploid tumour stemlines in 19 of 20 primary testicular cancers without significant difference of the ploidy values between seminomas and non-seminomas. In 7 of 8 analyzable histograms the S-phase activity was 22-51%. A metastatic mature teratoma had 6% cells in S-phase. These results support the clinical observation that testicular cancer is usually a rapidly growing human tumour. The high percentage of aneuploidy in testicular cancer may be of clinical value in the diagnosis of this malignancy.

Flow cytometric DNA measurements in paraffin-embedded bladder carcinoma tissue before and after precystectomy radiotherapy.

From 1976 through 1983, 48 patients with T2/T3 bladder carcinomas were treated with pre-operative radiotherapy (46 Gy) and total cystectomy. DNA histograms were recorded by flow cytometry (FCM) from all pre-treatment paraffin-embedded tumour biopsies and, if still present after radiotherapy, from tumour tissue from the cystectomy specimen. Five patients were excluded from the study because no DNA histograms could be recorded due to extensive destruction of the cell nuclei during preparation. Thus, 43 patients are completely evaluable. Before the start of radiotherapy, 32 bladder carcinomas were interpreted as non-diploid, whereas 11 were diploid. Non-diploidy of the pre-treatment biopsy was associated with radiotherapy-induced stage reduction (p = 0.13). The survival rates in patients with diploid and non-diploid tumours were 65% and 45% respectively at 4 years (p = 0.13). Although not statistically significant, our results suggest that DNA-FCM may provide clinically relevant information about the tumour biology and response to radiotherapy concerning bladder carcinomas.

Requirement of a reactive aldehyde moiety for aldehyde-mediated protection against cis-dichlorodiammineplatinum-induced cell inactivation.

The effect of the aromatic aldehydes benzaldehyde and salicylaldehyde, the glucose-acetal derivative 4,6-benzylidene-D-glucose (BG) and the glucoside salicylaldehyde-beta-D-glucoside (helicin) on cell inactivation induced by cis-dichlorodiammineplatinum (cis-DDP) was investigated using cultured human NHIK 3025 cells. Cell inactivation was measured as loss in the ability of single cells to give rise to macroscopic colonies following drug treatment. The fraction of cells surviving a 2 hr treatment with 10 microM cis-DDP increased from 0.012 +/- 0.004 to 0.10 +/- 0.03 when treatment was combined with at least 1 mM benzaldehyde or at least 0.2 mM salicylaldehyde. Of the two sugar-aldehyde derivatives only helicin protected cells from the inactivating effect of cis-DDP, although to a much lesser extent than either benzaldehyde or salicylaldehyde. While helicin retains the aldehyde moiety of salicylaldehyde, BG does not possess any free aldehyde group. Using synchronized cells we found these effects to appear in all phases of the cell cycle. Measurements of cell-associated platinum indicated that the degree of protection from the inactivating effects of cis-DDP by these aldehydes was related to the degree of reduced platinum accumulation. We conclude that this reduced accumulation may represent an inhibition of specific cell membrane uptake sites via Schiff based formation between membrane amino groups and aldehydes.

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.

DNA flow cytometric values in bladder carcinoma biopsies obtained from fresh and paraffin-embedded material.

DNA-histograms were obtained by flow cytometry (FCM) of nuclei prepared from fresh bladder carcinoma biopsies and from 100 micron sections of paraffin-embedded material from the same biopsies. Linear regression analysis of ploidy values from fresh material versus those from paraffin blocks showed excellent correlation (R2 = 0.957). Owing to the high background, the paraffin-embedded material was less suited for analysis of S-phase fraction. It is concluded that DNA FCM of nuclei obtained from paraffin-embedded bladder carcinoma biopsies yields reliable results concerning ploidy, but does not permit evaluation of the S-phase fraction.