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P L Olive

Publications and source records attributed to P L Olive.

At least 19 recordsLinked to original sources
PubMed2006

Endogenous expression of phosphorylated histone H2AX in tumors in relation to DNA double-strand breaks and genomic instability.

Microscopically visible gammaH2AX foci signify the presence of DNA double-strand breaks (dsbs) in irradiated cells. However, large foci are also observed in untreated tumour cells, and high numbers reduce the sensitivity for detecting drug or radiation-induced DNA breaks. SW756 cervical carcinoma cells that express about 50 gammaH2AX foci per cell (i.e., equivalent to the number of breaks produced by about 2Gy) showed similar numbers of dsbs as C33A cells that exhibit fewer than three foci per cell. The possibility that differences in numbers of these endogenous foci could be explained by genomic instability perhaps related to misrepair was examined. For 17cell lines selected from the panel of NCI-60 tumor cells previously characterized for karyotypic complexity [A.V. Roschke, G. Tonon, K.S. Gehlhaus, N. McTyre, K.J. Bussey, S. Lababidi, D.A. Scudiero, J.N. Weinstein, I.R. Kirsch, Karyotypic complexity of the NCI-60 drug-screening panel, Cancer Res. 63 (2003) 8634-8647], there was a significant trend (r=0.6) for cell lines with greater numbers of structural or numerical chromosomal rearrangements to show a higher background expression of gammaH2AX. Moreover, cells from this panel with wild-type p53 showed a significantly lower background level of gammaH2AX than cells with mutant p53. To confirm the importance of p53 expression, endogenous and radiation-induced gammaH2AX expression were analyzed using four isogenic SKOV3 cell lines varying in p53 function. Again, higher gammaH2AX expression was found in SKOV3 cell lines expressing mutant p53 compared to wild-type p53. HFL-1 primary lung fibroblasts showed a progressive increase in gammaH2AX as they moved towards senescence, confirming the importance of telomere instability in the development of at least some gammaH2AX foci. Therefore, the explanation for high endogenous levels of gammaH2AX in some tumor cells appears to be multifactorial and may be best described as a consequence of chromatin instability.

Cell Line, Tumor↗
PubMed2006

Comparison between pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix.

BACKGROUND: Although tumor hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is no consensus as to the method best suited for routine measurement. Binding of the chemical hypoxia marker, pimonidazole, and expression of the endogenous hypoxia markers HIF-1alpha and CAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advanced carcinoma of the cervix. METHODS: Two biopsies were taken one day after administration of pimonidazole and were analyzed for pimonidazole binding using flow cytometry or immunohistochemistry. CAIX and HIF-1alpha expression and degree of colocalization were measured in sequential antibody-stained sections. Patient subsets were examined for tumor oxygen tension using an Eppendorf electrode, S phase DNA content, or change in HIF-1alpha expression over the course of treatment. RESULTS: Approximately 6% of the tumor area stained positive for pimonidazole, HIF-1alpha, or CAIX. The CAIX positive fraction correlated with the pimonidazole positive fraction (r = 0.60). Weaker but significant correlations were observed between pimonidazole and HIF-1alpha (r = 0.31) and CAIX and HIF-1alpha (r = 0.41). Taking the extent of marker colocalization into consideration increased the confidence that all markers were identifying hypoxic regions. Over 65% of stained areas showed a high degree of colocalization with the other markers. Oxygen microelectrode measurements and S phase fraction were not correlated with the hypoxic fraction measured using the three hypoxia markers. HIF-1alpha levels tended to decrease with time after the start of therapy. CONCLUSIONS: Endogenous hypoxia marker binding shows reasonable agreement, in extent and location, with binding of pimonidazole. CAIX staining pattern is a better match to the pimonidazole staining pattern than is HIF-1alpha, and high CAIX expression in the absence (or low levels) of HIF-1alpha may indicate a different biology.

Antigens, Neoplasm↗
PubMed2005

Growth of V79 cells as xenograft tumors promotes multicellular resistance but does not increase spontaneous or radiation-induced mutant frequency.

A Chinese hamster V79 xenograft model was developed to determine whether cells subjected to a hypoxic tumor microenvironment would be more likely to undergo mutation at the HPRT locus. V79-171b cells stably transfected with VEGF and EGFP were grown subcutaneously in immunodeficient NOD/ SCID mice. V79-VE tumors were characterized for host cell infiltration, doubling time, hypoxic fraction, vascular perfusion, and response to ionizing radiation. When irradiated in vitro, the mutant frequency for a given surviving fraction did not differ for cells grown in vivo or in vitro. Similar results were obtained using HCT116 human colorectal carcinoma cells grown as xenografts. However, V79-VE cells grown as xenografts were significantly more resistant to killing than monolayers. The background mutant frequency and the radiation-induced mutant frequency did not differ for tumor cells close to or distant from blood vessels. Similarly, tumor cells from well-perfused regions showed the same rate of strand break rejoining and the same rate of loss of phosphorylated histone H2AX as cells sorted from poorly perfused regions. Therefore, deleterious effects of the tumor microenvironment on DNA repair efficiency or mutation induction could not be demonstrated in these tumors. Rather, development of multicellular resistance in V79-VE tumors acted to reduce mutant frequency for a given dose of radiation.

Animals↗
PubMed2003

Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays.

PURPOSE: Exposure to ionizing radiation results in phosphorylation of histone H2AX (gammaH2AX) at sites of DNA double-strand breaks. To determine the relationship between gammaH2AX formation and radiosensitivity, the rate of formation and loss of gammaH2AX were examined in several cultured cell lines following exposure to 253 kV X-rays. MATERIALS AND METHODS: Flow and image cytometry were both performed using a mouse monoclonal antibody against gammaH2AX. Immunoblotting was used to confirm cell line-dependent differences in antibody staining. Cell lines examined included V79 and CHO-K1 hamster cells, the human tumour cell lines SiHa, WiDr, DU145, WIL-2NS, HT144, HCC1937 and U87, and the normal cell strain HFL1. Radiosensitivity was measured using a standard clonogenic assay. RESULTS: Using flow cytometry, gammaH2AX formation was detected 1 h after doses as low as 20 cGy. Peak levels of gammaH2AX were observed within 15-30 min after irradiation and both the rate of radiation-induced gammaH2AX formation and loss were cell type dependent. Maximum levels of gammaH2AX formation were lower for HT144 cells mutant for the ataxia telangiectasia gene. Half-times of loss after irradiation ranged from 1.6 to 7.2 h and were associated with a decrease in the total number of foci per cell. The half-time of loss of gammaH2AX was correlated with clonogenic survival for 10 cell lines (r2=0.66). CONCLUSIONS: GammaH2AX can be detected with excellent sensitivity using both flow and image analysis. The rate of gammaH2AX loss may be an important factor in the response of cells to ionizing radiation, with more rapid loss and less retention associated with more radioresistant cell lines.

Animals↗
PubMed2002

Local hypoxia is produced at sites of intratumour injection.

Intratumour injection, commonly used for gene or drug delivery but also associated with needle biopsy or insertion of invasive measuring devices, may damage tumour microvessels. To examine this possibility, SCCVII tumours grown subcutaneously in C3H mice were injected with a 26 gauge needle containing 0.1 ml of the fluorescent dye Hoechst 33342 to label cells lining the track of the needle. Hoechst-labelled cells sorted from these tumours were more sensitive to killing by hypoxic cell cytotoxins (tirapazamine, RSU-1069) and less sensitive to damage by ionizing radiation. Hoechst-labelled cells also bound the hypoxia marker pimonidazole when given by i.p. injection. Intratumour injection transiently increased hypoxia from 18 to 70% in the tumour cells adjacent to the track of the needle. The half-time for return to pre-treatment oxygenation was about 30 min; oxygenation of tumour cells along the track had recovered by 20 h after intratumour injection. This effect could have significant implications for intratumour injection of drugs, cytokines or vectors that are affected by the oxygenation status of the tumour cells as well as potential effects on biodistribution via local microvasculature.

Animals↗
PubMed2002

Identification of genes differentially expressed in V79 cells grown as multicell spheroids.

PURPOSE: Growth of Chinese hamster V79 cells as multicell spheroids leads to an increase in resistance to killing by ionizing radiation and etoposide. Differential display was used to identify changes in gene expression that occur when cells are grown as spheroids. MATERIALS AND METHODS: Differential display was performed using exponentially growing Chinese hamster V79 cells and the outer cell layer of V79 spheroids. Using six different pairs of primers, 20 altered bands were selected. Eight genes, confirmed using reverse Northerns, showed a match in a GenBank search. Antibodies against a calcium-binding protein, mts1, confirmed differential expression of this protein. Intracellular free calcium levels were measured using fluo-3 fluorescence, and the effect of a calcium-binding agent on etoposide resistance was examined using the comet assay. RESULTS: Genes upregulated in the outer cell layer of spheroids relative to monolayers included: (1) mts1 (S100A4), a calcium binding protein implicated in proliferation, metastasis, cell adhesion, and angiogenesis; (2) cytochrome c oxidase II; (3) B-ind1, a mediator of Rac-1 signaling; (4) TRAM, an endoplasmic reticulum protein. Genes downregulated in spheroids were: (5) phosphoglycerate kinase; (6) ARL-3, a ras-related GTP binding protein; (7) MHC class III complement 4A; and (8) 2,4-dienoyl-CoA. Immunohistochemistry confirmed overexpression of mts1 and another calcium-binding protein, calreticulin, in V79 outer spheroid cells relative to monolayers. C6 rat glioma and SiHa human cervical carcinoma cells that demonstrate a contact effect also showed upregulation of mts1 or calreticulin, while WiDr colon carcinoma cells that lack contact resistance showed no change in expression of either calcium binding protein. Intracellular free calcium levels were found to be almost two times lower in the outer cells of V79 spheroids compared to monolayers. V79 monolayer and outer spheroid cells treated with the calcium chelating agent BAPTA-AM showed a similar level of DNA damage by etoposide. CONCLUSIONS: Expression of genes involved in calcium binding, signaling and metabolism are differentially expressed when V79 cells are grown as spheroids. Differences in the levels of intracellular calcium may underlie the contact effect.

Animals↗
PubMed2001

Carbonic anhydrase 9 as an endogenous marker for hypoxic cells in cervical cancer.

The presence of radiation-resistant hypoxic cells in some solid tumors is known to predict for relapse after radiotherapy. Use of an endogenous marker of hypoxia would be a convenient alternative to current methods that measure tumor oxygenation, provided the marker could be shown to reliably identify viable, radiation-resistant, hypoxic cells. Carbonic anhydrase 9 (CA9) is a transmembrane protein overexpressed in a wide variety of tumor types and induced by hypoxia. Using a monoclonal antibody and cell sorting, CA9-positive cells in SiHa cervical carcinoma xenografts growing in immunodeficient mice were found to be clonogenic, resistant to killing by ionizing radiation, and preferentially able to bind the hypoxia marker pimonidazole. CA9 and pimonidazole immunostaining were compared in formalin-fixed sections from tumors of 18 patients undergoing treatment for cancer of the cervix. Excellent colocalization was observed, although the area of the tumor section that bound anti-CA9 antibodies represented double the number of cells that bound anti-pimonidazole antibodies. Occasional regions staining with pimonidazole but not CA9 could be indicative of transient changes in tumor perfusion. Results support the hypothesis that CA9 is a useful endogenous marker of tumor hypoxia.

Animals↗
PubMed2001

A pilot study comparing intratumoral oxygenation using the comet assay following 2.5% and 5% carbogen and 100% oxygen.

PURPOSE: Tumor hypoxia has been purported to be an important biologic factor in the failure of radical radiotherapy to achieve local control in many tumor types. This study was designed to evaluate the effect of breathing high oxygen content gas mixtures (oxygen with 0%, 2.5%, or 5% carbon dioxide) on tumor oxygenation measured using the Eppendorf polarographic oxygen electrode and the comet assay in accessible, hypoxic human tumors. METHODS AND MATERIALS: Using Eppendorf pO2 histography to identify hypoxic tumors (median pO2 < or = 10 mmHg), eligible patients were systematically allocated either 100% oxygen (O2) or oxygen with 2.5% or 5% carbon dioxide (CO2). Tumors were treated with 6-10 Gy during which two fine needle aspirates (FNA) were obtained from different regions of the lesion, one at midway and the other at completion of the radiation exposure. Gas breathing was initiated 4 min before radiation was commenced. A 10-min interval was specified between the first and second halves of the radiation exposure to allow near maximal DNA repair prior to the second half of the radiation treatment. FNAs were performed within 2 min of cessation of radiation and the cells immediately suspended in buffered saline at 4 degrees C for analyses of hypoxic fraction using the comet assay. RESULTS: Fifteen evaluations were performed in 13 patients with hypoxic tumors (median O2 tension 2.75 mmHg) treated with a median dose of 8 Gy. The median hypoxic fraction determined using the comet assay fell from 0.36 to 0.13 (p = 0.001, Wilcoxon signed rank test) due to the addition of high oxygen content gases. CONCLUSIONS: In tumors defined as hypoxic using Eppendorf pO2 histography, a statistically significant reduction in the hypoxic fraction with the comet assay was found following administration of high oxygen content gases. These preliminary findings reveal a trend suggesting that 5% carbogen may reduce the hypoxic fraction by a greater margin than either 100% oxygen or 2.5% carbogen.

Analysis of Variance↗
PubMed2001

Measuring hypoxia in solid tumours--is there a gold standard?

Tumour hypoxia is known to be associated with aggressiveness and poor response to treatment, which has stimulated the development of several methods able to detect hypoxic tumours. To date, only one method, the oxygen microelectrode, has been used to provide pretreatment measures of tumour oxygenation that correlate with local control and disease-free survival. In an effort to validate new methods, comparisons have been made between the Eppendorf oxygen microelectrode, the comet assay, and hypoxia marker binding in tumours of patients undergoing curative treatment or palliative radiotherapy. These comparisons suggest that tumours with median oxygen tensions below 10 mmHg have relatively high hypoxic fractions as measured by the comet assay (> 0.20). The fraction of cells that binds pimonidazole, detected in cells obtained by fine-needle aspiration biopsy, correlates well with the hypoxic fraction measured using the comet assay. However, in general, hypoxic fractions measured by the comet assay and pimonidazole binding correlate only poorly with Eppendorf measurements performed for the same tumour. Factors that might be responsible for these differences, and problems associated with measuring the 'relevant' hypoxic population are discussed.

Biopsy, Needle↗
PubMed2001

Overnight lysis improves the efficiency of detection of DNA damage in the alkaline comet assay.

The ability to detect DNA damage using the alkaline comet assay depends on pH, lysis time and temperature during lysis. However, it is not known whether different lysis conditions identify different types of DNA damage or simply measure the same damage with different efficiencies. Results support the latter interpretation for radiation, but not for the alkylating agent MNNG. For X-ray-induced damage, cells showed the same amount of damage, regardless of lysis pH (12.3 compared to >13). However, increasing the duration of lysis at 5 degrees C from 1 h to more than 6 h increased the amount of DNA damage detected by almost twofold. Another twofold increase in apparent damage was observed by conducting lysis at room temperature (22 degrees C) for 6 h, but at the expense of a higher background level of DNA damage. The oxygen enhancement ratio and the rate of rejoining of single-strand breaks after irradiation were similar regardless of pH and lysis time, consistent with more efficient detection of strand breaks rather than detection of damage to the DNA bases. Conversely, after MNNG treatment, DNA damage was dependent on both lysis time and pH. With the higher-pH lysis, there was a reduction in the ratio of oxidative base damage to strand breaks as revealed using treatment with endonuclease III and formamidopyrimidine glycosylase. Therefore, our current results support the hypothesis that the increased sensitivity of longer lysis at higher pH for detecting radiation-induced DNA damage is due primarily to an increase in efficiency for detecting strand breaks, probably by allowing more time for DNA unwinding and diffusion before electrophoresis.

Animals↗
PubMed2001

RPA foci are associated with cell death after irradiation.

MacPhail, S. H. and Olive, P. L. RPA Foci are Associated with Cell Death after Irradiation. Radiat. Res. 155, 672-679 (2001). Complexes containing replication protein A (RPA) were observed in human TK6 and WIL-2NS lymphoblast cells and SiHa cervical carcinoma cells exposed to 250 kV X rays. Image analysis of individual cells with fluorescence-tagged anti-RPA antibodies was used to measure numbers of discrete foci per cell. RPA foci formed in S-phase cells in response to radiation doses as low as 0.5 Gy, and the number of foci/nucleus was linearly related to dose up to 50 Gy. The maximum number of cells with foci occurred 4-8 h after exposure to 4 Gy, and subsequently declined. However, the number of RPA foci per nucleus (in those cells with foci) reached a maximum after 2-4 h. Apoptotic nuclei from irradiated TK6 and WIL-2NS cells initially contained foci, but these were lost as degradation continued. Radiation-induced micronuclei in SiHa cells were greatly enriched for RPA foci, and cells with nuclei without foci often contained micronuclei with multiple RPA foci. In SiHa cells examined up to 7 days after 4 Gy, RPA foci reappeared in one or more cells in up to 90% of the surviving colonies, and some cells contained 150 or more distinct foci. Reappearance of these complexes could be indicative of radiation-induced genomic instability. These results are consistent with the idea that RPA foci observed several hours after irradiation represent irreparable lesions and as such might be useful in identifying radiosensitive cells.

Cell Death↗
PubMed2000

Changes in subcellular distribution of topoisomerase IIalpha correlate with etoposide resistance in multicell spheroids and xenograft tumors.

The outer cells of Chinese hamster V79 spheroids are about 10 times more resistant than monolayers to DNA damage and cell killing by the topoisomerase (topo) II inhibitor etoposide. Although the amount and catalytic activity of topo IIalpha are identical for monolayers or the outer cells of spheroids, and the cell proliferation rate is the same, our previous results indicated that phosphorylation of topo IIalpha is at least 10 times higher in V79 monolayers than in spheroids. Because phosphorylation of topo IIalpha has been associated with nuclear translocation, we examined subcellular distribution of Topo IIalpha in monolayers, spheroids, and xenograft tumors using immunohistochemistry. Topo IIalpha was located predominantly in the nucleus of V79, human SiHa, and rat C6 monolayers but was found mainly in the cytoplasm of the proliferating outer cells of spheroids formed from these cell lines. Conversely, the outer cells of WiDr human colon carcinoma spheroids showed predominantly nuclear localization of topo IIalpha, and only WiDr cells showed no increase in resistance to etoposide when grown as spheroids. Cells sorted from xenografts resembled the spheroids in terms of sensitivity to etoposide and location of topo IIalpha. When the outer cells of V79 spheroids were returned to monolayer growth, the rate of redistribution of topo IIalpha to the nucleus occurred with similar kinetics as the increase in sensitivity to killing by etoposide. Removal and return of individual outer V79 spheroid cells to suspension culture resulted in the translocation of topo IIalpha to the nucleus for the first 24 h, accompanied by an increase in sensitivity to DNA damage by etoposide. Therefore, the cytoplasmic topo IIalpha distribution in outer spheroid cells and tumors appears to correlate not with morphological changes associated with growth in suspension but rather with the presence of neighboring, noncycling cells.

Animals↗
PubMed2000

Vascular endothelial growth factor expression is independent of hypoxia in human malignant glioma spheroids and tumours.

We recently showed that severe hypoxia was not universally present adjacent to necrosis in human glioma xenografts and spheroids established from the M059K, M006, M006X, M006XLo and M010b cell lines. Using these glioma models, we wished to test whether oxygen serves as a regulator of cellular VEGF expression in situ. In situ hybridization (ISH) and immunohistochemistry (IHC) were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression in sections of glioma xenografts and spheroids in which hypoxic regions and regions with well-oxygenated necrosis were identified on contiguous sections by use of the hypoxia-specific marker, 3H-misonidazole. Independent validation of the presence of radiobiologically hypoxic cells in M006 xenografts was undertaken using the comet assay. Northern blotting analyses of monolayer cells demonstrated significant up-regulation of VEGF mRNA in the M006X line at oxygen concentrations of 6% and below. ISH analysis of VEGF mRNA showed unexpectedly strong staining for VEGF mRNA across the entire viable rim of M006X and M006XLo glioma spheroids. Similarly, in virtually all xenograft tumours of the M059K, M006 and M010b lines, VEGF ISH showed similar staining across all regions of healthy cells up to the border of necrosis. Only in one M006X tumour was there a suggestion of increased VEGF expression in cells adjacent to necrosis. IHC for VEGF showed good concordance with the ISH results. IHC analysis of the VEGF receptor flt-1 showed strong tumour cell staining in M006XLo glioma cells. In human glioma spheroids and xenograft tumours, regions of severe hypoxia do not correspond to areas of up-regulated VEGF expression; in fact, VEGF expression is quite uniform. Furthermore, this and our previous study demonstrate that levels of VEGF expression vary among sublines (M006, M006X and M006XLo) derived from a single human glioma specimen.

Base Sequence↗
PubMed2000

Comparison between the comet assay and pimonidazole binding for measuring tumour hypoxia.

Pimonidazole is finding increasing use in histochemical analyses of hypoxia in tumours. Whether it can identify every hypoxic cell in a tumour, and whether the usual subjective criteria used to define 'positive' cells are optimal, are less certain. Therefore, our aim was to develop an objective flow cytometry procedure for quantifying pimonidazole binding in tumours, and to validate this method by using a more direct indicator of radiobiologic hypoxia, the comet assay. SCCVII tumours in C3H mice were analysed for pimonidazole binding using flow cytometry and an iterative curve-fitting procedure, and the results were compared to the comet assay for the same cell suspensions. On average, cells defined as anoxic by flow analysis (n = 43 tumours) bound 10.8 +/- 0.95 times more antibody than aerobic cells. In samples containing known mixtures of aerobic and anoxic cells, hypoxic fractions as low as 0.5% could easily be detected. To assess the flow cytometry assay under a wider range of tumour oxygen contents, mice were injected with hydralazine to reduce tumour blood flow, or allowed to breathe various gas mixtures during the 90 min exposure to pimonidazole. Hypoxic fraction estimated by the pimonidazole binding method agreed well with the hypoxic fraction measured using the comet assay in SCCVII tumours (r2 = 0.87, slope = 0.98), with similar results in human U87 glioma cells and SiHa cervical carcinoma xenografts. We therefore conclude that this objective analysis of pimonidazole labelling by flow cytometry gives a convenient and accurate estimate of radiobiological hypoxia. Preliminary analyses of biopsies from 3 patients given 0.5 g m-2 pimonidazole also suggest the suitability of this approach for human tumours.

Animals↗
PubMed2000

Characterization of DNA-strand breakage induced in V79 cells by F 11782, a catalytic inhibitor of topoisomerases.

DNA damage induced in V79 cells by F 11782, or 2",3"-bis pentafluorophenoxyacetyl-4',6'-ethylidene-beta-D glucoside of 4'-phosphate-4'-demethylepipodophyllotoxin 2N-methyl glucamine salt, a novel dual catalytic inhibitor of topoisomerases I and II, was investigated using both alkaline and neutral versions of the comet assay methodology. A comparison was then made of the DNA damage induced by F 11782 with that induced by either etoposide or camptothecin under comparable experimental conditions. The results revealed that F 11782 initially induced less DNA strand breaks that either etoposide or camptothecin and rejoined such breaks more slowly. However, unlike these other drugs, the extent of DNA damage induced by F 11782 increased linearly with time of incubation. F 11782 produced both DNA single- and double-strand breaks without any clear specificity relative to phase of the cell cycle, although proliferating cells were preferentially damaged. The marked time-dependency of induction of DNA strand breaks by F 11782 may serve to explain, at least in part, its major in vivo antitumour activities.

Animals↗
PubMed1999

Increase in the fraction of necrotic, not apoptotic, cells in SiHa xenograft tumours shortly after irradiation.

BACKGROUND AND PURPOSE: Approximately 18% of the cells recovered by rapid mechanical dissociation of SiHa xenograft tumours contain large numbers of DNA strand breaks. The number of damaged cells increases to 30-40% 4-6 h after exposure to 5 or 15 Gy, returning to normal levels by 12 h. This observation is reminiscent of the rate of production of apoptotic cells in other murine and human xenograft tumours. The nature of this damage, rate of development and relation to cell proliferation rate were therefore examined in detail. MATERIALS AND METHODS: SiHa human cervical carcinoma cells were grown as xenograft tumours in SCID mice. Single-cell suspensions were prepared as a function of time after irradiation of the mouse and examined for DNA damage using the alkaline comet assay. Cell cycle progression was measured by flow cytometry evaluation of anti-bromodeoxyuridine-labelled tumour cells. RESULTS: Significant numbers of apoptotic cells could not be detected in irradiated SiHa tumours using an end-labelling assay, electron microscopy, or histological examination of thin sections. Instead, xenograft cells exhibiting extensive DNA damage in the comet assay were predominantly necrotic cells. The increase in the proportion of heavily damaged cells 4-6 h after irradiation could be the result of an interplay between several factors including loss of viable cells and change in production or loss of necrotic cells. Analysis of the progression of BrdUrd-labelled cells confirmed that while 35% of cells from untreated SiHa tumours had divided and entered G1 phase by 6 h after BrdUrd injection, none of the labelled cells from tumours exposed to 5 or 15 Gy had progressed to G1. CONCLUSIONS: The increase in the percentage of SiHa tumour cells with extensive DNA damage 4-6 h after irradiation is attributable to necrosis, not apoptosis. Cell cycle progression and cell loss are likely to influence the kinetics of appearance of both apoptotic and necrotic cells in irradiated tumours.

Animals↗