Erratum: Drug resistance in Chinese hamster ovary cells during recovery from severe hypoxia.
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Animals↗
Biomedical subjects
R M Sutherland
Publications and source records attributed to R M Sutherland.
Advances in simple immunoassays for decentralized testing.
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Chemistry, Clinical↗
Enhanced synthesis of stress proteins caused by hypoxia and relation to altered cell growth and metabolism.
Cultured cells maintained in very low oxygen levels alter their structure, metabolism and genetic expression. Culture conditions for cells were modified to minimise variation of nutrients and to allow normal survival levels after 24 h of hypoxic exposure. Under these hypoxic conditions, glucose consumption and lactate production rates were similar to aerobic rates until about 12 h after which the hypoxic rates increased. DNA and protein synthesis rates are continuously inhibited to about 48% or 55% of the respective aerobic rates. During this period of decreased protein synthesis, a set of proteins termed oxygen regulated proteins (ORPs), exhibits enhanced relative synthesis. The molecular weights of the five major ORPs are approximately 260, 150, 100, 80 and 33 kDa. While increased relative synthesis of oxygen regulated proteins is partly due to increased levels of mRNA which encode these proteins, the mechanism of enhanced synthesis of ORPs may be more complex.
Animals↗
Changes in growth characteristics and macromolecular synthesis on recovery from severe hypoxia.
Chinese hamster ovary cells subjected to severe hypoxia stop growing. When oxygen was reintroduced growth resumed, but at a slower rate. The longer the hypoxic stress, the slower the recovery growth rate. Six hours of hypoxia caused very little decrease in growth rate while a 24 h period almost halved the rate. Short hypoxic periods resulted in almost no growth lag, while longer periods caused significant lag. Clonogenic survival was 60% after 12 h of hypoxia and rose slowly during recovery, reaching control levels after 60 h. Following 24 h of hypoxia, survival remained around 60% throughout recovery. The cell cycle distribution after hypoxia was similar to that of aerobic cultures. After 4-6 h of recovery, a subpopulation of cells entered S phase, and reached G2 by 12 h. During this time few G2-M cells divided. With longer recovery, cells much larger than aerobic cells emerged, containing greater than 4C DNA content and enhanced amounts of RNA. When these cells were isolated, they exhibited slightly slower growth kinetics, greatly lengthened lag time and decreased survival when compared to aerobic cells or the smaller cells. Most of the extra DNA and RNA was lost within one cell cycle.
Animals↗
Modulators of tumor responsiveness to radioimmunotherapy.
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Adjuvants, Immunologic↗
Differentiation patterns in two- and three-dimensional culture systems of human squamous carcinoma cell lines.
Relative quantification of the pattern of differentiation of two squamous carcinoma cell lines of the female genital tract, A431 and CaSki, was studied in various experimental tissue culture states that are frequently used to evaluate drug and radiation effects on human tumors. Two- and three-dimensional in vitro cultures, ie, monolayers and multicellular tumor spheroids (MCTS), and nude mice-xenograft tumors as in vivo tumor models were compared. In addition, epidermal growth factor (EGF) was used comparatively in the in vitro studies. Morphologic signs of epithelial differentiation could be recognized in both cell lines gradually increasing from monolayers to MCTS to xenograft tumors. Cytokeratin (CK) expression is described as stable in A431 cells. Using immunohistochemistry, however, partial masking of CK antigens was found when applying the antibody 8.12 on monolayer cells and could be quantified by flow cytometric measurements. Fundamental cellular changes were found in a CaSki xenograft tumor, which showed newly established features of a keratinizing carcinoma after late onset of tumor growth. Epidermal growth factor caused reduction of both intercellular contacts and later onset of necrosis in MCTS, leading to an increased viability of the spheroids. Significant differences in differentiation of the tumor model systems indicates that the characterization of differentiation with immunohistochemistry and flow cytometry is necessary to assist interpretation of data obtained with these different tumor models.
Antibodies, Monoclonal↗
[Proliferation-associated antigens PCNA and Ki-67 in two- and three-dimensional experimental systems of human squamous epithelial carcinomas].
Multiparameter-flow-cytometry was used to compare the levels of two proliferation-associated antigens, proliferating cell nuclear antigen (PCNA) and the Ki-67-related antigen, in squamous carcinoma cells grown as monolayers, multicellular spheroids (MCTS), and xenograft tumors. While the level of Ki-67-positive cells decreased with time of culture, the percentage of PCNA-positive cells stayed high in all experimental states investigated (up to 5 weeks of growth). The reduction of the mean fluorescence of PCNA/cell indicated a different way of regulation of this antigen in squamous carcinoma cells in comparison to other cell types.
Animals↗
Heterogeneity of glutathione content in human ovarian cancer.
Intracellular glutathione (GSH) has been shown to be one of the major factors modulating tumor response to a variety of commonly used anti-neoplastic agents. In this study the GSH contents of human ovarian tumors from primary biopsies, nude mouse xenografts, and in vitro cell cultures were compared. Pronounced intratumor cell-to-cell heterogeneity in GSH content was observed in primary patient biopsies when assessed using flow cytometry. For example, in an ascites biopsy from a newly diagnosed patient, a 5.6-fold difference in GSH concentration existed between the cell subpopulations with the 5% highest and 5% lowest GSH contents. Similar intratumor heterogeneity in GSH content was also evident in nude mouse xenografts. In addition, for a particular tumor line, the intertumor variations of GSH content among individual whole tumors were much less than the intratumor variation among slices from an individual tumor. Nude mouse xenografts of human ovarian cancer had GSH contents that were on average only slightly lower (30%) than those found in primary biopsies. In contrast, tumor cells grown as in vitro cultures, particularly those in exponential growth phase, had GSH contents considerably greater (1.3- to 3.5-fold) than those found in situ. Plateau phase cultures, however, had lower GSH contents and were more comparable to those observed in tumors in vivo. Overall, it may be concluded that in situations where GSH plays an important part in determining tumor response to a particular treatment, nude mouse xenografts may represent the most appropriate experimental model system.
Animals↗
Drug resistance in Chinese hamster ovary cells during recovery from severe hypoxia.
Chinese hamster ovary cells exposed to hypoxia developed an 80-fold resistance to a subsequent 1-hour exposure to doxorubicin (ADR) in air. Recovery in air before drug exposure resulted in loss of resistance. Cells exposed to hypoxia for 20 hours followed by a 15-hour recovery were still twofold to threefold more resistant than aerobic cells to a short pulse of ADR. A subpopulation of cells was generated that was at least twice as large as aerobic cells and contained greater than normal G2-M DNA content. This subpopulation showed no resistance to a continuous exposure to either ADR or methotrexate, nor was it more resistant to a pulse of ADR than the remaining cells with normal DNA content. Our data indicate that hypoxia can produce significant ADR resistance. However, conditions resulting in overproduced DNA did not cause significant additional resistance.
Animals↗
Enhancement of sensitivity of human squamous carcinoma cells to radiation by epidermal growth factor.
Experiments were done to determine the effect of murine epidermal growth factor (EGF) on the radiation responses of the human squamous carcinoma cell line CaSki grown as an exponential monolayer culture. The radiation responses studied included recovery from potentially lethal damage (PLDR) and recovery from sublethal damage (SLDR). The presence of EGF in the cell culture either after irradiation (during the clonogenic assay period) or continuously before, during, and after irradiation enhanced the radiosensitivity of the cells and reduced their plating efficiency (PE). However, these effects were not as great when EGF was present in the cell culture only before and during irradiation. This enhancement of radiosensitivity was associated with a reduction in the shoulder region of the cell survival curves. The PE reduction and radiosensitivity enhancement were maximum with 10 ng of EGF/mL. However, EGF present continuously in the cell culture, including during the 6-hour repair period, had no effect on cellular PLDR or SLDR. The single-stranded DNA breaks present in large numbers in cells immediately after irradiation returned to control levels by 6 hours. Moreover, EGF present in cell cultures for the 48 hours before irradiation and during the 6-hour repair period had no effect on the DNA alkaline elution profiles of either control or irradiated cells. EGF did not affect the growth of unirradiated cells; however, it extended the lag-phase period for growth of irradiated cells. In summary, this EGF-induced radiosensitivity enhancement was not correlated with the effects of EGF on PE, cell growth, PLDR, or SLDR.
Carcinoma, Squamous Cell↗
131I-anticarcinoembryonic antigen therapy of LS174T human colon adenocarcinoma spheroids.
LS174T human colon adenocarcinoma multicell spheroids were used to study the radiobiological aspects of radioimmunotherapy. The spheroids were incubated in 131I-anticarcinoembryonic antigen (B7) at an antibody concentration of 0.5 microgram/ml and at 131I concentrations of 2.5 and 7.5 microCi/ml. After incubation times of 90 h, clonogenic cells per spheroid were reduced by 1400-fold and 23-fold at the high and low 131I concentrations, respectively. 131I Nonspecific antibody (PX63) resulted in 2- and 1.2-fold reductions. Spheroid diameter was not significantly affected by therapy but histological examination revealed that there had been a significant reduction in the cell density, particularly near the spheroid surface. Using a theoretical model to estimate radiation dose, a radiation survival curve was constructed. The resulting curve was somewhat concave suggesting the presence of a resistant population of cells. It is likely that this observation is primarily due to the fact that the inner cells received a lower dose than the outer cells. A population of radiobiologically hypoxic cells in the inner portion of the spheroids may also have contributed to the decreasing slope of the curve as well as ongoing cell division leading to new cells which receive a lower radiation dose per cell cycle. Because of the ability to estimate radiation dose for a given biological effect, these types of experiments may allow predictions of the efficacy of radiolabeled antibody therapy for micrometastatic disease.
Adenocarcinoma↗
The relationship between radiation response of human squamous carcinoma cells and specific metabolic changes induced by chronic hypoxia.
Chronic hypoxia can cause a range of metabolic changes within cells, such as increasing the synthesis rate of a group of proteins, oxygen regulated proteins (ORPs) and reducing glutathione (GSH) content. We are currently interested in the radiation responses of cells during recovery after chronic hypoxia in relation to these metabolic changes. Experiments have therefore been carried out on the influence of hypoxic pretreatment to gamma rays of two human squamous carcinoma cell lines, A431 (vulva) and CaSki (cervix), in exponential and plateau growth phases. In both cell lines, the synthesis rate of ORPs reached to a maximum level after 12 hr of hypoxia and during the same time, the cellular GSH content reduced by about 50%, but both returned to the control levels by 12 hr of reoxygenation. After 12 hr of hypoxic incubation at 37 degrees C, cells were allowed to reoxygenate in air for 10 min (on ice) or 12 hr at 37 degrees C before irradiation. Clonogenic assays were performed immediately after irradiation. Compared to the aerobic control, the radiosensitivity of both cell lines reoxygenated for 10 min after hypoxia increased significantly and later returned to the aerobic control level by 12 hr of reoxygenation. Since aerobic A431 cells treated with 10 mM buthionine sulfoximine for 12 hr did not increase the radiosensitivity, the enhanced aerobic radiosensitivity observed after chronic hypoxia was unlikely to be directly related to decreased GSH content. Further investigations of ORPs and other associated metabolic changes caused by chronic hypoxia have been in progress to determine their possible role in this enhanced radiation sensitivity.
Carcinoma, Squamous Cell↗
31P NMR spectroscopy and HbO2 cryospectrophotometry in prediction of tumor radioresistance caused by hypoxia.
The aim of this study was to search for possible relationships between the fraction of radiobiologically hypoxic cells in tumors and their 31P NMR spectral parameters and intracapillary HbO2 saturations. Four different tumor lines, two murine sarcomas (KHT, RIF-1) and two human ovarian carcinoma xenografts (MLS, OWI), were used. When tumor volume increased from about 200 mm3 to about 2000 mm3, hypoxic fraction increased from 12 to 23% for the KHT line, from 0.9 to 1.7% for the RIF-1 line, and from 9 to 28% for the MLS line. The OWI line showed similar hypoxic fractions at 200 (17%) and 2000 mm3 (15%). Tumor bioenergetic status decreased, that is, the inorganic phosphate (Pi) resonance increased and the phosphocreatine (PCr) and nucleoside triphosphate beta (NTP beta) resonances decreased, with increasing tumor volume for the KHT, RIF-1, and MLS lines, whereas the OWI line did not show any changes in the 31P NMR spectral parameters during tumor growth. Similarly, tumor HbO2 saturation status, that is, the fraction of vessels with HbO2 saturation above 30%, decreased with increasing tumor volume for the KHT, RIF-1, and MLS lines, but remained unchanged during tumor growth for the OWI line. Although the data indicated a relationship between hypoxic fraction and tumor bioenergetic status as well as tumor HbO2 saturation status within a specific line during tumor growth, there was no correlation between hypoxic fraction and tumor bioenergetic status or tumor HbO2 saturation status across the four tumor lines. This may have occurred because cell survival time under hypoxic stress as well as fraction of non-clonogenic, but metabolically active hypoxic cells differed among the tumor lines. This indicates that 31P NMR spectroscopy and HbO2 cryospectrophotometry data have to be supplemented with other data to be useful in prediction of tumor radioresistance caused by hypoxia.
Animals↗
Enhanced synthesis of specific proteins, RNA, and DNA caused by hypoxia and reoxygenation.
When Chinese Hamster ovary cells were exposed to severe hypoxia they responded with an enhanced synthesis rate of a small group of specific proteins, which we have termed the oxygen regulated proteins (ORPs). The five major ORPs had molecular weights of 33, 80, 100, 150, and 260 kD. ORP260 was the first ORP to be synthesized at enhanced rates after the induction of hypoxia, reaching its maximal synthesis rate after only a few hours. The other ORPs responded slower, but were being synthesized at maximal rates by 8 hr of hypoxia. Upon reoxygenation, the synthesis rates of the ORPs declined rapidly with ORP260 again responding most rapidly. They all reached control levels by 12 hr of recovery. The induction of enhanced synthesis of these proteins required a very severe degree of hypoxia. They were not seen at oxygen levels higher than 0.03%. While enhanced synthesis of the ORPs was turned on and off quickly with induction of, and recovery from hypoxia, the proteins were not degraded at high rates once synthesized. All five major ORPs have apparent half-lives in excess of 24 hr. When cells were exposed to hypoxia for 20 hr and then allowed to recover for 15 hr, a population of cells was generated that had, on average, at least twice the cell volume as aerobic control cells. These large cells also contained greater than 4C DNA content as well as enhanced amounts of RNA.
Animals↗
Changes in cellular glutathione content during adriamycin treatment in human ovarian cancer--a possible indicator of chemosensitivity.
Patients with ovarian cancer often respond well to combination chemotherapy initially but the majority eventually relapse when, with further treatment, the initially successful regimen proves ineffectual. The cause of such failures frequently has been attributed to the development of drug resistance. Although the mechanisms of acquired resistance in situ are still poorly understood, studies in vitro have shown that cells selected for resistance to one drug often exhibit cross-resistance to other seemingly unrelated agents, suggesting a somewhat generalised mechanism of resistance. We have studied the role of glutathione (GSH) and drug transport in determining the sensitivity to adriamycin (ADR) of a panel of human ovarian cell lines established directly from biopsies of patients with diverse treatment histories. These cell lines exhibited inherent differences in sensitivity to ADR by a dose factor of up to 3; a difference that was considerably less than what has been reported when cells were selected for drug resistance in vitro. The differences in drug sensitivity reported here among the various cell lines appeared to be unrelated to drug transport, in terms of both influx and efflux. Moreover, although these cell lines have a wide range of GSH content, there was only a poor correlation between drug sensitivity and cellular GSH content per se. However, when exposed to a clinically relevant dose of ADR, the GSH content of cell lines that were 'sensitive' decreased, whereas that of cell lines that were 'resistant' increased. To take these time-dependent changes in GSH into consideration, the area under the GSH content versus time curve (AUC), with and without ADR treatment, was calculated for each cell line. When this latter factor was included in the analysis, greatly improved correlations were found between GSH kinetic parameters and responses to ADR. In particular, ADR resistance was found to be closely correlated with the positive changes in absolute GSH AUC following ADR treatment (r = 0.92; P less than 0.01). Using 35S-labelled cysteine and methionine as tracers, it was found that the essential difference between the 'resistant' and 'sensitive' lines was that the 'resistant' lines had higher steady-state rates of GSH synthesis than the 'sensitive' lines. These results demonstrate that changes in cellular GSH concentration during treatment may be an important indicator of tumour cell response to ADR.
Adenocarcinoma↗
Growth and radiation sensitivity of the MLS human ovarian carcinoma cell line grown as multicellular spheroids and xenografted tumours.
The growth characteristics and the radiation sensitivity of multicellular spheroids of the MLS human ovarian carcinoma cell line grown in spinner culture in atmospheres of 5% CO2 in air or 5% CO2, 5% O2 and 90% N2 were studied and compared to that of MLS xenografted tumours. The spheroids grew exponentially with a volume-doubling time of approximately 24 h up to a diameter of approximately 580 microns and then the growth rate tapered off, more for spheroids grown at the low than at the high oxygen tension. Thirty days after initiation, the spheroid diameters were approximately 1,500 microns at the low and 2,100 microns at the high oxygen tension. The tumour volume-doubling times were approximately 8 days (V less than 200 mm3) and 17 days (V = 1,000-4,000 mm3). The histological appearance of the spheroids and the tumours was remarkably similar; both developed large central necrosis and both were composed of epithelial cells and showed pseudoglandular structures with lumen. The spheroids were slightly less differentiated than the tumours. The intrinsic, cellular radiation sensitivity was independent of whether the cells were grown in vitro as spheroids or in vivo as tumours, as revealed by irradiating single cells from dissociated spheroids and tumours under aerobic conditions and intact spheroids and tumours under hypoxic conditions. Studies of 1,600 microns spheroids grown in 5% CO2 in air showed that the intrinsic radiation sensitivity of the chronically hypoxic cells was the same as that of acutely hypoxic cells. The fraction of radiobiologically hypoxic cells under these conditions was approximately 15% and similar to those of 9% (V = 200 mm3) and 28% (V = 2,000 mm3) found for the tumours. Spheroids with diameter of 1,200 microns did not show survival curves parallel to those for acutely hypoxic cells, i.e. they did not contain a measurable fraction of clonogenic cells at complete radiobiological hypoxia. The final portion of their survival curves represented partially hypoxic cells; the OERs were 1.6 and 1.3 for spheroids grown at the high and the low oxygen tension, respectively. The considerable similarity between the spheroids and the tumours suggests that MLS spheroids constitute a valuable in vitro model for studies of human tumour radiation biology and related physiological processes. MLS spheroids may be particularly useful in studies of therapeutic consequences of partial radiobiological hypoxia since complete hypoxia and different levels of partial hypoxia can be studied separately by varying spheroid size and the oxygen tension in the culture medium.
Cell Line↗
Indirect presentation of alloantigen in vivo.
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Animals↗
The radiation response of cells recovering after chronic hypoxia.
Experiments were performed to study the influence of hypoxic pretreatment on the radiation response of A431 human squamous carcinoma cells. Reaeration for 10 min after chronic hypoxia (greater than 2 h) was found to enhance the radiosensitivity of A431 cells, and the maximal effect was seen for those cells reaerated after 12 h of hypoxia. The radiosensitivity enhancement for reaerated cells after 12 h of hypoxia was maximized by 5 min after the return to aerobic conditions and reached the control level by 12 h of reaeration. This enhanced radiosensitive state was characterized by a reduced shoulder region and increased slope of the radiation dose-response curve for cells in both the exponential and plateau phases of growth. There was a slight increase in the number of G1 and decrease in the number of S and G2 + M cells for both exponential- and plateau-phase cultures following 12 h hypoxic treatment. Although growth inhibition induced by 12 h of hypoxia was seen for cells in the exponential phase, there was no cell number change in the plateau-phase culture after hypoxia. Plating efficiency (PE) of cells in both growth phases was reduced by 30% after hypoxia. Furthermore, in the exponential-phase culture, the extent of reduction in PE after hypoxia was similar among cells in different phases of the cell cycle. Although S-phase cells in exponentially growing cultures were relatively more resistant to radiation than G1 and G2 + M cells, the cell age-response pattern was the same whether the cells had been aerobic or hypoxic before reaeration and irradiation. Furthermore, the enhancement ratio associated with reaeration after 12 h of hypoxia for these three subpopulations of cells was 1.3. Our results indicate that the increase in radiosensitivity due to reaeration after chronic hypoxia is unlikely to be related to the changes of cell cycle stage and growth phase during hypoxic treatment.
Aerobiosis↗