Repair, redistribution and repopulation in V79 spheroids during multifraction irradiation

Abstract. Development of predictive assays for measuring tumour radiosensitivity has generated much recent interest, particularly with the recognition that tumour cell survival at doses of about 2 Gy may correlate well with tumour curability. Clinical data, however, suggest that overall treatment time may be of considerable significance in radioresponsive tumours, especially for rapidly growing tumours capable of accelerated repopulation. Because neither factor can be repeatedly assessed in human tumours, we used cells growing as multicell spheroids to determine whether the initial radiation response would be predictive for multifraction exposures, or whether other factors including repopulation rate should be considered. Potential problems of hypoxia and reoxygenation were avoided by using small spheroids which had not yet developed radiobiologically hypoxic regions. Repair and redistribution dominated the responses in the first two or three exposures, with repopulation playing a minor role. As the fractionation schedule was extended, however, repopulation between fractions largely determined the number of viable cells per spheroid. We conclude that the radiation response of cells from untreated spheroids provides a general indication of net sensitivity, but that repair and redistribution produces considerable variation in radiosensitivity throughout a fractionation protocol. Ultimately, repopulation effects may dominate the multifraction response.     

Human DNA polymerase epsilon is expressed during cell proliferation in a manner characteristic of replicative DNA polymerases.

In order to shed light on the role of mammalian DNA polymerase epsilon we studied the expression of mRNA for the human enzyme during cell proliferation and during the cell cycle. Steady-state levels of mRNA encoding DNA polymerase epsilon were elevated dramatically when quiescent (G0) cells were stimulated to proliferate (G1/S) in a similar manner to those of DNA polymerase alpha. Message levels of DNA polymerase beta were unchanged in similar experiments. The concentration of immunoreactive DNA polymerase epsilon was also much higher in extracts from proliferating tissues than in those from non-proliferating or slowly proliferating tissues. The level of DNA polymerase epsilon mRNA in actively cycling cells synchronized with nocodazole and in cells fractionated by counterflow centrifugal elutriation showed weaker variation, being at its highest at the G1/S stage boundary. The results presented strongly suggest that mammalian DNA polymerase epsilon is involved in the replication of chromosomal DNA and/or in a repair process that may be substantially activated during the replication of chromosomal DNA. A hypothetical role for DNA polymerase epsilon in a repair process coupled to replication is discussed.     

Proliferative changes in two murine tumours in response to single or fractionated doses of X-rays

Abstract. Studies were carried out to investigate proliferative changes in two murine experimental tumours in response to radiation. Results were generated using bro-modeoxyuridine labelling and flow cytometry. This study demonstrates the possible ambiguity of previous studies using tritiated thymidine in which inability to discriminate normal and tumour cell components in murine tumours may lead to different values for cell kinetic parameters. In particular, the sarcoma F appeared to have a growth fraction of 0.62 when all cells were considered; in reality the growth fraction of the tumour cells only (based on DNA content discrimination) was close to unity. Radiation, administered either as single or fractionated doses, caused little change in the proliferative characteristics of the sarcoma F tumour but had profound effects on the adenocarcinoma Rhodesia tumour. Major changes were the accumulation of cells in G₂ for several days after the end of the radiation treatment in both tumours and a dramatic drop in labelling index of the Rhodesia tumour. In neither tumour was there any evidence to suggest an increase in tumour cell proliferation during or after the irradiations. The diploid cells within the sarcoma F tumour showed an initial depression of labelling index followed by a rapid increase overshooting the control labelling index at higher radiation doses. Much of the effects could be attributed to cell cycle delays.     

Experimental results and clinical implications of the four R's in fractionated radiotherapy

Summary Experimental and clinical data on the four R's in fractionated radiotherapy are reviewed. The clinical importance of redistribution has not been proven in the experiment yet. On reoxygenation no unequivocal data in human cancer exists and a lot of variability in rodent tumours. Repair and regeneration are the most important of the four R's in fractionated radiotherapy. The presented experimental and clinical evidence suggests a differential response between tumour and late responding normal tissues with regard to these two R's. Tumours appear to have, in general, a smaller capacity for repairing sublethal radiation damage but a higher capacity for repopulation than late responding normal tissues.     

Modelling the Cell Cycle and Cell Movement in Multicellular Tumour Spheroids

This paper analyses a recent mathematical model of avascular tumour spheroid growth which accounts for both cell cycle dynamics and chemotactic driven cell movement. The model considers cells to exist in one of two compartments: proliferating and quiescent, as well as accounting for necrosis and apoptosis. One particular focus of this paper is the behaviour created when proliferating and quiescent cells have different chemotactic responses to an extracellular nutrient supply. Two very different steady-state behaviours are identified corresponding to those cases where proliferating cells move either more quickly or more slowly than quiescent cells in response to a gradient in the extracellular nutrient supply. The case where proliferating cells move more rapidly leads to the commonly accepted spheroid structure of a thin layer of proliferating cells surrounding an inner quiescent core. In the case where proliferating cells move more slowly than quiescent cells the model predicts an interesting structure of a thin layer of quiescent cells surrounding an inner core of proliferating and quiescent cells. The sensitivity of this tumour structure to the cell cycle model parameters is also discussed. In particular variations in the steady-state size of the tumour and the types of transient behaviour are explored. The model reveals interesting transient behaviour with sharply delineated regions of proliferating and quiescent cells.     

Experimental chemotherapy and concepts related to the cell cycle

Scheduling of chemotherapy is limited by damage to normal tissues, and tolerated schedules are dependent on normal tissue recovery. Most anticancer drugs are more toxic to proliferating cells and the fall and recovery of granulocyte counts after chemotherapy may be explained by the effect of drugs on rapidly proliferating precursor cells in the bone marrow. It is argued that serious toxicity due to myelosuppression most often occurs because of damage to proliferating precursors that may be recognized in bone marrow rather than to stem cells. In contrast, therapy that is aimed at producing cure or long-term remission of tumours must be directed at killing tumour stem cells. The evidence that tumours contain a limited population of cells which can repopulate the tumour after treatment (and are therefore tumour stem cells) is reviewed critically. While there is quite strong evidence for a limited population of target cells, evidence from studies on metastases suggests that the tumour cells which may express this stem cell property may change with time. The stem cell concept has major implications for predictive assays. Although colony-forming assays appear to have a sound biological background for predicting tumour response, technical problems prevent them from being used routinely in patient management. Cells in tumours are known to be heterogeneous and at least three types of heterogeneity may influence tumour response to drug treatment: the development of subclones with differing properties including drug resistance; variation in cellular properties due to differentiation during clonal expansion; and variation in properties due to nutritional status and micro-anatomy. Heterogeneity in drug distribution within solid tumours may occur because of limited drug penetration from blood vessels, and nutrient-deprived cells in solid tumours may be expected to escape the toxicity of some anticancer drugs as well as being resistant to radiation because of hypoxia. This may occur both because nutrient-deprived cells have a low rate of cell proliferation, and also because of poor drug penetration to them. There is a need for improved understanding of the mechanisms that lead to cell death in tumours. If these mechanisms were understood, it might be possible to simulate them by therapeutic manoeuvres. Recent research from our laboratory suggests that the combination of low extracellular pH and hypoxia may be very toxic to cells in nutrient-deprived regions. Drugs which limit the cell's ability to survive in regions of acid pH may provide strategy for therapy of nutrient-deprived cells.     

The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation.

The radiosensitivity of spermatogonial stem cells to X rays was determined in the various stages of the cycle of the seminiferous epithelium of the CBA mouse. The numbers of undifferentiated spermatogonia present 10 days after graded doses of X rays (0.5-8.0 Gy) were taken as a measure of stem cell survival. Dose-response relationships were generated for each stage of the epithelial cycle by counting spermatogonial numbers and also by using the repopulation index method. Spermatogonial stem cells were found to be most sensitive to X rays during quiescence (stages IV-VII) and most resistant during active proliferation (stages IX-II). The D0 for X rays varied from 1.0 Gy for quiescent spermatogonial stem cells to 2.4 Gy for actively proliferating stem cells. In most epithelial stages the dose-response curves showed no shoulder in the low-dose region.     

Differential sensitivities to gamma radiation of human bladder and testicular tumour cell lines

Gamma radiation sensitivities of continuous cell lines from nine human tumours were measured, comparing four derived from transitional cell carcinomas of the bladder with five from non-seminomatous germ cell tumours of the testis. The testicular cells were significantly more radiosensitive than the bladder cells, corresponding to the response to therapy of these tumour types in patients. These observations indicate that radiosensitivity is retained in vitro and is an inherent property of the testicular tumour cells. These gamma radiation sensitivities were compared with those of SV40-transformed fibroblasts derived from a normal individual and one with the heritable disease, ataxia-telangiectasia (A-T). The bladder cells had gamma radiation sensitivities similar to that of the SV40-transformed normal line. The testicular cells were hypersensitive to gamma radiation, although not as sensitive as the SV40-transformed A-T line. A-T cells, unlike those derived from normal individuals, continue to synthesize DNA at a normal rate following radiation exposure, prompting a comparison of the kinetics of DNA synthesis in three bladder and three testicular tumour cell lines. One of the bladder and two testicular lines showed a reduced inhibition when compared to the other tumour cell lines and the SV40-transformed normal line. Thus there was no clear association between DNA synthesis inhibition and radiosensitivity.     

Effect of hypothermia on radiosensitization

Hypothermia reduces metabolism and oxygen utilization by tissues. If the blood supply to a solid tumour can be maintained at a sufficient level, the hypoxic fraction of tumour cells may be reduced and radiosensitivity increased. This may be achieved if hyperbaric oxygen is used in combination with the hypothermia. The blood supply and oxygen tension have been measured in C3H mouse mammary tumours under hypothermia and hyperbaric oxygen, and the enhancement of radiosensitivity by hyperbaric oxygen has been estimated in mice irradiated at different temperatures with and without anaesthesia. Measurement of xenon-133 clearance showed that the blood supply of a tumour tended to increase when anaesthetized mice became hypothermic. Oxygen cathode data showed that the oxygen tension tended to be relatively higher in tumours and lower in subcutaneous tissue when mice exposed to hyperbaric oxygen became hypothermic under anaesthesia. Hyperbaric oxygen enhanced the radiation response of the tumour in terms of an increase in regrowth delay by a factor of 1.7 when the mice had been anaesthetized, whether or not they became hypothermic. A lower factor of 1.4 was obtained without anaesthesia although induced hypothermia increased the response to a small extent. We conclude that anaesthesia and hypothermia affect oxygen metabolism in tumours by different mechanisms.     

Flow-cytometric identification and follow-up of mice exposed to x-irradiation: Evaluation of a model system

An experimental model system is presented that allows the identification and follow-up of mice exposed to ionizing radiation using flow-cytometric measurements of peripheral blood cells. In an experiment, properties of peripheral blood cells were analysed with flow cytometry for a rapid identification of individuals exposed to radiation. Individuals were then followed longitudinally in an attempt to identify those developing neoplasias. Male CBA mice, 25 days old, were subjected to fractionated x-irradiation (4 × 1.31 Gy) to induce haematopoietic malignancies. By repeated blood sampling followed by flow cytometry, frequencies of micronucleated erythrocytes and of proliferating nucleated cells were determined. Neoplasias were diagnosed by histopathology. Five days after the end of radiation exposure, increased frequencies of proliferating cells, polychromatic erythrocytes and micronucleated normochromatic erythrocytes clearly distinguished the exposed group from the control group. Increased cell proliferation in peripheral blood cells could be used to identify animals with manifest tumours, although these animals were at a late stage of tumour development. Animals with thymic lymphoma (not generalized) could not be identified with the flow-cytometric parameters used. We consider that this model system has a potential use when a small number of risk individuals need to be identified and monitored within a large population.     

Accumulation of DNA damage and cell death after fractionated irradiation

The purpose of this work was to determine how fractionated radiation used in the treatment of tumors affects the ability of cancer as well as normal cells to repair induced DNA double-strand breaks (DSBs) and how cells that have lost this ability die. Lymphocytic leukemia cells (MOLT4) were used as an experimental model, and the results were compared to those for normal cell types. The results show that cancer and normal cells were mostly unable to repair all DSBs before the next radiation dose induced new DNA damage. Accumulation of DSBs was observed in normal human fibroblasts and healthy lymphocytes irradiated in vitro after the second radiation dose. The lymphocytic leukemia cells irradiated with 4 × 1 Gy and a single dose of 4 Gy had very similar survival; however, there was a big difference between human fibroblasts irradiated with 4 × 1.5 Gy and a single dose of 6 Gy. These results suggest that exponentially growing lymphocytic leukemia cells, similar to rapidly proliferating tumors, are not very sensitive to fraction size, in contrast to the more slowly growing fibroblasts and most late-responding (radiation therapy dose-limiting) normal tissues, which have a low proliferation index.     

PROLIFERATION STUDIES OF THE ENDOTHELIAL AND SMOOTH MUSCLE CELLS OF THE MOUSE MESENTERY AFTER IRRADIATION

A continuous labelling technique was employed to study the effects of external β-radiation on the proliferation of endothelial cells and smooth muscle cells in the mesenteric arterioles of mice. Labelled and non-labelled cells of either type were determined by autoradiographic techniques in control animals and at different times (3, 12 and 48 weeks) after single doses of 20 and 45 Gy (2000 and 4500 rads). The fraction of cells labelled, even after 7 days of repeated injections was very low in all instances. Calculations showed very long turnover times for the two cell populations in control animals (>2 years for endothelium and >3 years for smooth muscle). After 20 and 45 Gy, no significant increase in endothelial proliferation was seen except at 3 weeks. No significant increase in labelling was observed in smooth muscle at any time after irradiation. These labelling data have been compared with the pattern of cell depletion of the irradiated endothelium. It was concluded that the depletion was much earlier than expected for a slowly proliferating tissue, if all the cells were cycling very slowly. Such an early depletion is, however, consistent with cell death resulting from a small proportion of the cells having a short cell cycle. The recovery of the endothelial cell numbers between 9 and 12 months was not accompanied by a rise in the fraction of labelled cells. It is suggested that repopulation may occur from outside the treated area.     

Considerations in the design of possible cell cycle effective drugs

Antineoplastic agents are known to induce differential cytotoxic and cytostatic effects throughout the cell cycle. Many drugs have greater toxicity for cycling cells and act selectively at one or more phases of the cycle and may cause partial synchrony of surviving cells. However, these observations have been generally carried out on in vitro systems only and present a variety of complexities and pitfalls. Furthermore, human tumours are often characterized by a relatively low fraction of proliferating cells and present a large cellular heterogeneity as far as their cytogenetic, cytokinetic, and clonogenic features and their responses to drugs are concerned. Therefore, resistance to chemotherapy is due to various factors characterizing, in some instances, each individual tumour. In spite of the advent of technological advances such as flow cytometry, it is still difficult to design kinetic-orientated therapies especially for the treatment of solid tumours. Consequently, it is also difficult to design protocols based on cell cycle effective drugs. The possibility remains, at least for the moment, to stratify tumours according to their cellular heterogeneity. Different protocols could then be assigned to classes of tumours. Such an approach could be completed by further advances in the cellular monitoring of individual tumours.     

The effect of alterations in haematocrit on tumour sensitivity to X-rays

Hypoxic cells in human tumours probably contribute to the failure of radiotherapy in some sites. Changes in the oxygen carrying capacity of the blood, such as in anaemia, have been shown to influence tumour response. The effect of acute and chronic changes in haematocrit on the radiosensitivity of three mouse tumours (EMT6, KHT and RIF-1) were studied. Alterations in haematocrit were achieved by bleeding followed by retransfusion. When radiation was preceded immediately by an acute reduction in haematocrit (anaemia), radiosensitivity was markedly reduced in each tumour. An acute rise in haematocrit (polycythaemia) increased or decreased X-ray sensitivity depending on its severity. The optimum haematocrit for maximum sensitivity was always found to be at a level 5-10 per cent above normal. When the time between induction of anaemia and irradiation was increased, simulating a progressively longer duration of anaemia, marked changes in radiosensitivity of all the tumours were observed. A short duration of anaemia resulted in a resistant tumour with each cell line, but the resistance was gradually lost as the anaemia was prolonged, even though no recovery in haematocrit occurred. The rate of recovery to normal radiosensitivity varied from 24 to 72 hours in the different tumours. Therefore, only haematocrit changes which occurred within 1-3 days of a dose of radiation affect the radiosensitivity of these tumours.     

The Effect of X-Irradiation On Cell Loss In Five Solid Murine Tumours, As Determined By the 125Iudr Method

The rate of cell loss in irradiated RIF-1, EMT6, KHJJ, B16 and KHT tumours was studied using the ¹²⁵IUdR loss technique. Administration of ¹²⁵IUdR preceded localized tumour irradiation by 2 days. Loss of tumour radioactivity was measured for 6–8 days after irradiation. the blood flow to some tumours was occluded during, and for 30 min following, injection of the label to measure the amount of radioactivity entering the tumour as a result of reutilization of label from the gut epithelia and influx of labelled host cells. Irradiation did not significantly alter the amount of radioactivity entering these clamped tumours during the 8–10 days after injection of ¹²⁵IUdR. This permitted comparison of irradiated and control groups based on the loss of radioactivity from the non-occluded tumours. Irradiation of RIF-1, EMT6, KHJJ or B16 tumours with doses of 600, 1400, 2400 or 4400 rads produced no significant increase in the rate of loss of tumour radioactivity. This suggested that, in the population of labelled cells, cell lysis following irradiation proceeded slowly. In contrast, KHT tumours showed a significant increase in loss rate following each radiation dose, although the increase was dose-independent. In all tumour systems, the constant rate of cell loss after radiation appeared to coincide with published reports of tumour growth responses after irradiation. the present data suggest that the manner of expression of radiation-induced cell killing results from the cellular proliferative status, i.e. whether a cell is cycling or non-cycling.     

Cell proliferation kinetics of normal and tumour tissue in vitro: quiescent reproductive cells and the cycling reproductive fraction

Current methods for measuring the cell kinetics of human tumours are made and interpreted within the context of a simplistic two compartment model for cell proliferation, consisting of cells that are cycling and those that are not. It is now recognized that the non-cycling compartment of many tumours is heterogeneous, composed of non-reproductive end-stage cells and reproductive cells that are dormant/quiescent. We have developed an in vitro analysis that distinguishes for the first time quiescent reproductive cells from non-reproductive end-stage cells and have integrated this analysis with monolayer clonogenic and suicide assays to simultaneously quantitate the duration of the cell cycle and reproductive cells that are: cycling, quiescent, clonogenic, and non-reproductive end-stage cells. We have defined a new parameter, the Cycling Reproductive Fraction (CRF), which is the cycling cell population referenced specifically to the reproductive cell population. Measurements of CRF from 72 tumour biopsies and from 5 normal foreskins showed that CRF approached 100% in some tumours; however, CRF showed near normal values (< 1%) in others suggesting that cell cycle control may be maintained in some tumours. Because of CRF's improved specificity, we believe that CRF may enhance classification, prognostication, and the optimization and prediction of response to chemotherapy.     

Cyclin D1 overexpression perturbs DNA replication and induces replication-associated DNA double-strand breaks in acquired radioresistant cells

Fractionated radiotherapy (RT) is widely used in cancer treatment, because it preserves normal tissues. However, repopulation of radioresistant tumors during fractionated RT limits the efficacy of RT. We recently demonstrated that a moderate level of long-term fractionated radiation confers acquired radioresistance to tumor cells, which is caused by DNA-PK/AKT/GSK3β-mediated cyclin D1 overexpression. The resulting cyclin D1 overexpression leads to forced progression of the cell cycle to S-phase, concomitant with induction of DNA double-strand breaks (DSBs). In this study, we investigated the molecular mechanisms underlying cyclin D1 overexpression-induced DSBs during DNA replication in acquired radioresistant cells. DNA fiber data demonstrated that replication forks progressed slowly in acquired radioresistant cells compared with corresponding parental cells in HepG2 and HeLa cell lines. Slowly progressing replication forks were also observed in HepG2 and HeLa cells that overexpressed a nondegradable cyclin D1 mutant. We also found that knockdown of Mus81endonuclease, which is responsible for resolving aberrant replication forks, suppressed DSB formation in acquired radioresistant cells. Consequently, Mus81 created DSBs to remove aberrant replication forks in response to replication perturbation triggered by cyclin D1 overexpression. After treating cells with a specific inhibitor for DNA-PK or ATM, apoptosis rates increased in acquired radioresistant cells but not in parental cells by inhibiting the DNA damage response to cyclin D1-mediated DSBs. This suggested that these inhibitors might eradicate acquired radioresistant cells and improve fractionated RT outcomes.     

PROLIFERATION KINETICS OF THE MOUSE BLADDER AFTER IRRADIATION

The proliferative response of the mouse bladder was investigated, using continuous labelling with tritiated thymidine, at various times after a single dose of radiation. Bladder epithelial and vascular endothelial cells were studied. The cell turnover rate in unirradiated epithelium and endothelium was found to be extremely slow (in excess of 1 year). Irradiation with a single dose of 25 Gy resulted in compensatory proliferation of the epithelium but the response was not initiated for many months. At 3 months after irradiation there was little difference from the control proliferation rate, but from 6 to 22 months after irradiation (the end of the study) there was a period of sustained rapid proliferation with the cell turnover time reduced to approximately 1 week. The increase in proliferative activity observed at 22 months was found to be dose—dependent. Endothelial cells in the blood vessels of the submucosa also showed an increased turnover rate after irradiation and the timing of this reponse was found to be similar to that of the epithelium. The onset of compensatory proliferation in both cell types was found to coincide with marked histological and functional changes in the bladder. In this slowly proliferating tissue, the onset of rapid compensatory proliferation after irradiation is delayed and occurs at the time that functional impairment is observed. This supports the postulate that proliferation is unlikely to contribute much to the sparing effect of prolonged fractionated radiotherapy in slowly dividing tissues.     

Sensitivity of human glioma U-373MG cells to radiation and the protein kinase C inhibitor, calphostin C

We assessed the radiosensitivity of the grade III human glioma cell line U-373MG by investigating the effects of radiation and the specific protein kinase C inhibitor, calphostin C on the cell cycle and cell proliferation. Irradiated glioma U-373MG cells progressed through G1-S and underwent an arrest in G2-M phase. The radiosensitivity of U-373MG cells to graded doses of either photons or electrons was determine by microculture tetrazolium assay. The data was fitted to the linear-quadratic model. The proliferation curves demonstrated that U-373MG cells appear to be highly radiation resistant since 8 Gy was required to achieve 50% cell mortality. Compared to radiation alone, exposure to calphostin C (250 nM) 1 h prior to radiation decreased the proliferation of U-373MG by 76% and calphostin C provoked a weakly synergistic effect in concert with radiation. Depending on the time of application following radiation, calphostin C produced an additive or less than additive effect on cell proliferation. We postulate that the enhanced radiosensitivity observed when cells are exposed to calphostin C prior to radiation may be due to direct or indirect inhibition of protein kinase C isozymes required for cell cycle progression.     

Comparative investigations on the effect of different dose schedules of the phase-specific drug vincristine (VCR) on the proliferation kinetics of a solid experimental tumour

The aim of the present investigation was to study the effect of a high bolus injection (1 X 2.1 mg) of vincristine (VCR) during the phase of tumour growth retardation at the 14th day after transplantation and to compare the findings with the results of single (1 X 0.35 mg) and repeated (6 X 0.35 mg) applications of this cytostatic drug. Furthermore, an attempt was made to induce a synchronization of tumour cell proliferation. It was found that the effect on the volume growth was very pronounced after the high bolus injection and the repeated application of vincristine compared with the single low dose of the cytostatic drug. A synchronization of the tumour cell proliferation by flow out of the mitotic block could not be demonstrated. On the other hand a modest simultaneous recruitment of previously non-cycling tumour cells into the cell cycle occurred in the periphery of the tumour after the high bolus injection. The repeated application and the high bolus injection of VCR increased the cytostatic effect, especially in the tumour centre, related to the more slowly proliferating tumour cell compartment.