Cell population kinetics of the rhabdomyosarcoma R1H of the rat after single doses of X-rays

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H of the rat following local irradiation with single subcurative X-ray doses of 7.5, 15 and 30 Gy was studied. Several parameters were sequentially measured over a time interval of 4 weeks after irradiation: the ratio of the number of tumour to host cells, and the cellular DNA content of tumour and host cells, were determined by flow cytometry; the amount of DNA per gram of tumour tissue was determined biochemically; the clonogenic fraction of tumour cells was obtained from in vitro colony assay; and the tumour volume was assessed by in situ caliper measurements. From the amount of DNA per gram and the average DNA content per cell, the total number of cells per gram of tumour tissue was obtained. From this and the other parameters measured, the number of clonogenic tumour cells, non-clonogenic tumour cells and nucleated host cells per tumour, as well as their variation with time and dose, could be derived. The results showed that there was a lag period prior to depopulation amounting to 3.8 +/- 1.4, 1.4 +/- 0.8 or 0 +/- 0.7 days for 7.5, 15 or 30 Gy, respectively. The rate of depopulation of non-clonogenic tumour cells increased with dose; the halving times of non-clonogens were 4.7 +/- 1.8, 2.6 +/- 0.7 or 2.1 +/- 0.4 days for the three doses applied. There were no indications that proliferation of doomed cells contributed significantly to tumour growth after irradiation. After lag periods that were similar in length to those prior to depopulation, a massive immigration of host cells was observed. Under certain conditions more than 97 per cent of the cells present in irradiated tumours were found to be of host origin. There was a lag period before the onset of repopulation by clonogenic tumour cells, the length of which increased from 2.7 +/- 0.7 to 5.0 +/- 0.8 or 6.3 +/- 1.0 days for 7.5, 15 or 30 Gy, respectively. The initial rate of repopulation increased with radiation dose; after the end of the lag period the doubling time of clonogenic tumour cells (in controls amounting to 3.7 +/- 0.2 days) was 3.1 +/- 0.1, 2.1 +/- 0.1 and 1.1 +/- 0.1 days for the three doses applied.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics of depopulation, repopulation and host cell infiltration in the rhabdomyosarcoma R1H after 14 MeV neutron irradiation.

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H in the rat was studied following irradiation with 5 Gy of 14 MeV neutrons. Several parameters were sequentially measured over a time period of 4 weeks after irradiation: the tumour volume was assessed by in situ caliper measurements; the numerical density of tumour cells was obtained by morphometry; the clonogenic fraction of tumour cells was derived from in vitro colony assay; and the numerical ratio of host to tumour cells was determined by flow cytometry. From these primary parameters the number of clonogenic tumour cells, non-clonogenic tumour cells, and nucleated host cells per tumour, as well as their variation with time, were derived. The results were compared with two sets of data obtained previously for the same tumour exposed to 15 Gy of 200 kVp X-rays. Survival of tumour cells was reduced to 5.5 +/- 0.5% by 5 Gy neutrons and to 4.5 +/- 0.5% by 15 Gy X-rays, i.e. an RBE of close to 3. There was a lag period before the onset of repopulation (4.9 +/- 0.4 days and 4.9 +/- 0.5 days, respectively), followed by a high initial rate of repopulation corresponding to a doubling time of 2.0 +/- 0.2 days for neutrons and 2.1 +/- 0.2 days for X-rays. The rate of depopulation was significantly different for the two treatment modalities; the halving time for the number of non-clonogenic tumour cells was 11 +/- 4 days for neutrons and 2.8 +/- 0.5 days for X-rays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in epidermal radiosensitivity with time associated with increased colony numbers

Epidermal clonogenic cell survival and colony formation following irradiation were investigated and related to radiosensitivity. A rapid in vivo/in vitro assay was developed for the quantification of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. Bromodeoxyuridine (BrdU)-labelled cells in full thickness epidermal sheets were visualized using standard immunohistochemistry. In unirradiated skin, approximately 900 BrdU-positive cells mm(-2) were counted. In a time sequence experiment, BrdU-positive cell numbers increased from an average of 900 cells mm(-2) to approximately 1400 cells mm(-2) after BrdU-labelling for 2-24 h. In irradiated skin, colonies containing >/=16 BrdU-positive cells were seen for the first time at days 14/15 after irradiation. The number of these colonies per cm(2) as a function of skin surface dose yielded a cell survival curve with a D(0)-value (+/-SE) of 3.9+/-0.6 Gy. This relatively high D(0)-value is possibly due to a rapid fall off in depth dose distribution for the iridium-192 source and consequently a substantial contribution of hair follicular epithelium to colony formation. At 14/15 days after irradiation, the ED(50) level of 33.6 Gy for the in vivo response of moist desquamation corresponded with 2.7 colonies cm(-2). Surprisingly, the number of colonies increased with time after irradiation with an estimated doubling time of approximately 4 days, while the D(0)-value remained virtually unchanged. This increase in colony numbers could be due to migration of clonogenic cells, to the recruitment of dormant clonogenic cell survivors by elevated levels of cytokines, or to both. Although frequent biopsying caused increased cytokine levels, which had a systemic effect on unirradiated skin, it had no influence on colony formation in irradiated skin. Smaller colonies, containing 4-8 cells or 9-15 cells, were abundant, particularly after higher doses, which resulted in higher D(0)-values. The majority of these small colonies were abortive and did not progress to larger colonies. There was no statistical evidence for significant variations in the interanimal responses.     

The temporal and spatial changes in cell proliferation within the irradiated crypts of the murine small intestine

The detailed temporal and spatial changes in the labelling index in crypts of the small intestine of the mouse have been analysed after 8.0 Gy gamma-irradiation. The labelling index was determined for each cell position in the crypts at 34 different times between 3 and 192 h after irradiation. The changes between consecutive time points have been analysed to determine the details of the crypt shrinkage and crypt repopulation phenomena. The following points can be made: (1) There is a dramatic reduction in the overall labelling of the crypt which begins within 3 h and is at its minimum by 15 h postirradiation. Most of this shrinkage can be attributed to continued near-normal emigration of cells from the crypt to the villus while mitosis is reduced or absent, and a possibly premature maturation within the transit population. (2) The labelling index never falls below 34 per cent of control, i.e. many labelled cells persist and continue to replicate their DNA at all times postirradiation. (3) Repopulation begins in the lower regions of the crypt. The first changes are an increase in labelling at cell positions 3-8 that begins at 3 h and reaches a peak at 12 h. There is a second increase in proliferation at the crypt base that begins at about 15 h and reaches a peak at 22-32 h postirradiation. There is a third peak which begins at about 46 h and reaches a peak at 60-70 h. (4) There is a reduction in proliferation at the crypt base that begins at about 72 h postirradiation. (5) The mid and upper crypt population shrinks initially to reach a minimum at about 15 h, after which there is a steady increase to reach a peak at about 72 h. The labelling spreads into the crypt-villus boundary area beginning at about 32 h. There is a reduction of proliferative activity in the mid-crypt region that begins at about 72 h. (6) There is a dramatic overshoot in overall labelling index at 72 h, which involves mainly the upper crypt. This does not revert to normal levels within the 192 h time scale of the present experiments. There is a mild overshoot in labelling at the crypt base at 48-78 h with a return to normal levels thereafter.(ABSTRACT TRUNCATED AT 400 WORDS)     

The circadian rhythm for the number and sensitivity of radiation-induced apoptosis in the crypts of mouse small intestine

Apoptosis is a mode of cell death involving nuclear pycnosis, cytoplasmic condensation and karyorrhexis. Changes in the number of apoptotic cells at various times (3-12 h) after a single dose of either 0.5 or 9.0 Gy given at 09.00, 21.00 or 03.00 h were studied in histological sections of small intestinal crypts of mice. The incidences of apoptosis were examined 3 or 6 h after irradiation at different times of day with different doses of gamma-rays ranging from 0.15 to 9.0 Gy. Survival curves were constructed from the dose-incidence curves for apoptosis, using the number of apoptotic cells after high doses (NM) as the maximum cell population size. The mean lethal doses (Do) for the dose range 0-0.5 Gy were calculated for each time of day. A circadian rhythm in both Do and NM values was detected, indicating that both the number and sensitivity of radiation-induced apoptosis were changing throughout the day. A possible explanation based on the cell-cycle states of the target cell population for apoptosis (presumably functional stem cells) was drawn. Most of the target cells were assumed to be in an extended G1 phase. Around 21.00 h a transition from G1 to S phase takes place in some of these cells (approximately seven or eight cells per whole crypt). The S phase then lasts till around 06.00 h. They may be at G2 and M around 06.00-09.00 h, and then they re-enter G1. The circadian rhythm for the number and sensitivity of the cells susceptible to apoptosis obtained in the present report agrees well with this pattern of cell-cycle phases of target cells.     

Radiobiology and Stathmokinetics of Intestinal Crypts Associated with Patches of Peyer

Summary

The stathmokinetics and radiobiology of intestinal crypts directly adjoining the lymphoid patches of Peyer, have been compared with those of non-patch-associated crypts. Patch crypts contain an additional one to two rings of cells, the Mitotic Index for the whole crypt is higher than in non-patch crypts, and the apparent cell cycle time is insignificantly lower. Using single and split doses of gamma-rays, dose—survival curves were obtained for whole intestinal crypts, from which single-cell survival curves were derived for the clonogenic cells of the crypt. For a single-hit, multitarget, model, the extrapolation numbers of the cell survival curves for patch and non-patch crypts were the same (≈ 35) but the final D0 for cells of the patch crypts was significantly higher (2·1 versus 1·7 Gy). A linear-quadratic fit gave a similar ratio of α/β (≈ 10) for the two curves. For a given level of crypt depletion, the number of clonogenic cells per crypt derived by the use of equal split doses of radiation, was the same for patch and non-patch crypts. This number is a function of the dose regime employed: the higher the level of crypt depletion, the higher the derived number of cells (range 10 to 45, for non-patch crypts).     

Determination of colony numbers in pig epidermis as an estimate for radiosensitivity. A rapid assay based on in vitro BrdU-labelling

A rapid assay has been developed for the quantitation of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. The number of surviving clonogenic cells per unit area was related to the epidermal in vivo response of moist desquamation. After irradiation with single doses, ranging from 20 to 36 Gy, skin biopsies were taken and incubated in dispase for enzymatic separation of the epidermis and dermis. Full thickness epidermal sheets were labelled with bromodeoxyuridine (BrdU) in vitro. Proliferating cells were visualized using standard immunohistochemical procedures. Cell groups containing > or = 16 cells were counted as colonies. These colonies were first seen on day 14/15 after irradiation. The number of colonies per cm2, as a function of skin surface dose, yielded a cell survival curve with a D0 (+/- SE) of 3.87 +/- 0.57 Gy. The ED50 for the epidermal in vivo reaction of moist desquamation corresponded with a colony density of 2.7 colonies per cm2. After higher doses, abundant smaller colonies of 4-8 BrdU-positive cells were seen and these were more radioresistant, as represented by higher D0 values.     

Cell Population Kinetics of the Rhabdomyosarcoma R1H of the Rat after Single Doses of X-rays

Summary

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H of the rat following local irradiation with single subcurative X-ray doses of 7·5, 15 and 30 Gy was studied. Several parameters were sequentially measured over a time interval of 4 weeks after irradiation: the ratio of the number of tumour to host cells, and the cellular DNA content of tumour and host cells, were determined by flow cytometry; the amount of DNA per gram of tumour tissue was determined biochemically; the clonogenic fraction of tumour cells was obtained from in vitro colony assay; and the tumour volume was assessed by in situ caliper measurements. From the amount of DNA per gram and the average DNA content per cell, the total number of cells per gram of tumour tissue was obtained. From this and the other parameters measured, the number of clonogenic tumour cells, non-clonogenic tumour cells and nucleated host cells per tumour, as well as their variation with time and dose, could be derived. The results showed that there was a lag period prior to depopulation amounting to 3·8 ± 1·4, 1·4 ± 0·8 or 0 ± 0·7 days for 7·5, 15 or 30 Gy, respectively. The rate of depopulation of non-clonogenic tumour cells increased with dose; the halving times of non-clonogens were 4·7 ± 1·8, 2·6 ± 0·7 or 2·1 ± 0·4 days for the three doses applied. There were no indications that proliferation of doomed cells contributed significantly to tumour growth after irradiation. After lag periods that were similar in length to those prior to depopulation, a massive immigration of host cells was observed. Under certain conditions more than 97 per cent of the cells present in irradiated tumours were found to be of host origin. There was a lag period before the onset of repopulation by clonogenic tumour cells, the length of which increased from 2·7 ± 0·7 to 5·0 ± 0·8 or 6·3 ± 1·0 days for 7·5, 15 or 30 Gy, respectively. The initial rate of repopulation increased with radiation dose; after the end of the lag period the doubling time of clonogenic tumour cells (in controls amounting to 3·7 ± 0·2 days) was 3·1 ± 0·1, 2·1 ± 0·1 and 1·1 ± 0·1 days for the three doses applied. Nevertheless, all repopulation curves could be described by one particular Gompertz function (whose parameters also give a good fit to the volume growth of the undisturbed tumour), indicating that the rate of repopulation corresponds to the growth rate of untreated tumours that contain a comparable number of clonogens as are left in the irradiated tumours.     

Impact of increased cell loss on the repopulation rate during fractionated irradiation in human FaDu squamous cell carcinoma growing in nude mice

PURPOSE: To determine the impact of increased necrotic cell loss on the repopulation rate of clonogenic cells during fractionated irradiation in human FaDu squamous cell carcinoma in nude mice. MATERIALS AND METHODS: FaDu tumours were transplanted into pre-irradiated subcutaneous tissues. This manoeuvre has previously been shown to result in a clear-cut tumour bed effect, i.e. tumours grow at a slower rate compared with control tumours. This tumour bed effect was caused by an increased necrotic cell loss with a constant cell production rate. After increasing numbers of 3-Gy fractions (time intervals 24 or 48 h), graded top-up doses were given to determine the dose required to control 50% of the tumours (TCD50). All irradiations were given under clamp hypoxia. RESULTS: With increasing numbers of daily fractions, the top-up TCD50 decreased from 37.9 Gy (95% CI: 31; 45) after single dose irradiation to 14.1 Gy (8; 20) after irradiation with 15 fractions in 15 days. Irradiation with 18 daily 3-Gy fractions controlled more than 50% of the tumours without a top-up dose. After irradiation with six fractions every second day, the top-up TCD50 decreased to 26.9 Gy (22; 32). No further decrease of the TCD50 was observed after 12 and 18 irradiations every second day. Assuming a constant increase of TCD50 with time, the calculated doubling time of the clonogenic tumour cells (Tclon) was 7.8 days (4.4; 11.3). The Tclon calculated for FaDu tumours growing in pre-irradiated tissues was significantly longer (p=0.0004) than the Tclon of 5.1 days (3.7; 6.5) determined under the same assumptions in a previous study for FaDu tumours growing in normal subcutaneous tissues. CONCLUSIONS: Increased necrotic cell loss by pre-irradiation of the tumour bed resulted in longer clonogen doubling times during fractionated radiotherapy of human FaDu squamous cell carcinoma. This implies that a decreased necrotic cell loss might be the link between reoxygenation and repopulation demonstrated previously in the same tumour model.     

Diary of events

Summary

The detailed temporal and spatial changes in the labelling index in crypts of the small intestine of the mouse have been analysed after 8·0 Gy γ-irradiation. The labelling index was determined for each cell position in the crypts at 34 different times between 3 and 192 h after irradiation. The changes between consecutive time points have been analysed to determine the details of the crypt shrinkage and crypt repopulation phenomena. The following points can be made: (1) There is a dramatic reduction in the overall labelling of the crypt which begins within 3 h and is at its minimum by 15 h postirradiation. Most of this shrinkage can be attributed to continued near-normal emigration of cells from the crypt to the villus while mitosis is reduced or absent, and a possibly premature maturation within the transit population. (2) The labelling index never falls below 34 per cent of control, i.e. many labelled cells persist and continue to replicate their DNA at all times postirradiation. (3) Repopulation begins in the lower regions of the crypt. The first changes are an increase in labelling at cell positions 3–8 that begins at 3 h and reaches a peak at 12 h. There is a second increase in proliferation at the crypt base that begins at about 15 h and reaches a peak at 22–32 h postirradiation. There is a third peak which begins at about 46 h and reaches a peak at 60–70 h. (4) There is a reduction in proliferation at the crypt base that begins at about 72 h postirradiation. (5) The mid and upper crypt population shrinks initially to reach a minimum at about 15 h, after which there is a steady increase to reach a peak at about 72 h. The labelling spreads into the crypt-villus boundary area beginning at about 32 h. There is a reduction of proliferative activity in the mid-crypt region that begins at about 72 h. (6) There is a dramatic overshoot in overall labelling index at 72 h, which involves mainly the upper crypt. This does not revert to normal levels within the 192 h time scale of the present experiments. There is a mild overshoot in labelling at the crypt base at 48–78 h with a return to normal levels thereafter. It can be concluded that early changes in the crypt, probably the small levels of acute cell death, result in an early stimulation of stem cell proliferation, the signals for which must be local in action. These in turn lead to further rounds of stem cell proliferation and transit cell repopulation.     

Selective inhibition of the epidermal growth factor receptor tyrosine kinase by BIBX1382BS and the improvement of growth delay, but not local control, after fractionated irradiation in human FaDu squamous cell carcinoma in the nude mouse

PURPOSE: To investigate the effect of BIBX1382BS, an inhibitor of the epidermal growth factor receptor tyrosine kinase, on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumour growth and local tumour control after fractionated irradiation over 6 weeks in nude mice. FaDu human squamous cell carcinoma is epidermal growth factor receptor positive and significant repopulation during fractionated irradiation was demonstrated in previous experiments. MATERIALS AND METHODS: Receptor status, receptor phosphorylation, cell cycle distribution, cell proliferation and clonogenic cell survival after irradiation were assayed with and without BIBX1382BS (5 microM) in vitro. Tumour volume doubling time, BrdUrd and Ki67 labelling indices and apoptosis were investigated in unirradiated tumours growing in NMRI nude mice treated daily with BIBX1382BS (50 mg kg(-1) body weight orally) or carrier. Tumour growth delay and dose-response curves for local tumour control were determined after irradiation with 30 fractions within 6 weeks. RESULTS: BIBX1382BS blocked radiation-induced phosphorylation of the epidermal growth factor receptor and reduced the doubling time of FaDu cells growing in vitro by a factor of 4.9 (p=0.008). Radiosensitivity in vitro remained unchanged after incubation with BIBX1382BS for 3 days and decreased moderately after 6 days (p=0.001). BIBX1382BS significantly reduced the volume doubling time of established FaDu tumours in nude mice by factors of 2.6 when given over 15 days (p<0.001) and 3.7 when applied over 6 weeks (p<0.001). When given simultaneously to fractionated irradiation, growth delay was significantly prolonged by an average of 33 days (p=0.003). Local tumour control was not improved by BIBX1382BS. The radiation doses necessary to control 50% of the tumours locally were 63.6 Gy (95% confidence interval 55; 73) for irradiation alone and 67.8 Gy (60; 77) for the combined treatment (p=0.5). CONCLUSIONS: Despite clear antiproliferative activity in rapidly repopulating FaDu human squamous cell carcinoma and significantly increased tumour growth delay when combined with fractionated irradiation, local tumour control was not improved by BIBX1382BS. The results do not disprove that epidermal growth factor receptor inhibition might enhance the results of radiotherapy. However, the results imply that further preclinical investigations using relevant treatment schedules and appropriate endpoints are necessary to explore the mechanisms of action and efficacy of such combinations.     

Growth kinetics of C3H10T1/2 cells exposed to low-LET radiation

Growth curves and size of the colonies of C3H10T1/2 cells exposed to low-LET radiation (31 MeV protons) were determined after 0, 1, 3, 5, and 7 Gy. The data show that: cell density at confluence was 3.3 x 10(4) cells/cm2; the initial division delay was very small; in the first 15 h the increase in the cell number was essentially the same at all doses; at 100 h the colony size distribution was very large, ranging from 0 to 7 generations, even within the control population. The temporal dependence of the growth properties of surviving and non-surviving cells was represented by the equation N = N0(Fe(a(t - dD] + (1 - F)ea/bD(1 - e - bD(1 - e - bD(t - dD]) where F is the surviving fraction, t the time of sampling, a the growth rate, d the division delay per unit dose, b the rate per unit of dose at which the non-surviving cells lose their ability to divide. The resulting values were: a = 0.029 +/- 0.002 h-1; b = 0.0041 +/- 0.0009 h-1 Gy-1 and d = 1 +/- 0.8 h Gy-1. It was found that growth curves are affected by non-surviving progeny up to 150, 200 and 250 h after irradiation at 3, 5 and 7 Gy, whereas at longer times the population consists essentially of progeny of surviving cells.     

Differentiation status of human squamous cell carcinoma xenografts does not appear to correlate with the repopulation capacity of clonogenic tumour cells during fractionated irradiation

PURPOSE: To investigate the magnitude and kinetics of repopulation in a moderately well differentiated UT-SCC-14 human squamous cell carcinoma [hSCC] in nude mice. This question is of interest because clinical data indicate a higher repopulation capacity in those SCC that have preserved characteristics of differentiation, which appears to be in contrast to results on FaDu and GL hSCC previously reported from this laboratory. METHODS AND MATERIALS: UT-SCC-14 tumours were transplanted subcutaneously into the right hind leg of NMRI nu/nu mice. Fractionated radiation treatments were delivered, either under clamped hypoxia at 5.4 Gy/fraction or under ambient conditions (consistent with an OER of 2.7). Tumours were irradiated every day, every 2nd day, or every 3rd day with 6, 12 or 18 fractions. 1, 2 or 3 days after the last fraction, graded top-up-doses under clamped conditions were given for the purpose of estimating the 50% tumour control dose (TCD50). A total of 22 TCD50 assays were performed and analysed using maximum likelihood techniques. RESULTS: The data demonstrate a slow but significant repopulation of clonogenic cells during fractionated irradiation of UT-SCC-14 hSCC. The results under hypoxic conditions are consistent with a constant repopulation rate, with a clonogenic doubling time (Tclon) of 15.6 days (95% CI: 9.7, 21.4). This contrasts with ambient conditions where Tclon was 68.5 days (95% CI: 124, 161). Both Tclon values are longer than the 6-day volume doubling time of untreated tumours. CONCLUSIONS: Less pronounced repopulation for irradiation under ambient compared to clamped hypoxic conditions might be explained by preferential survival of hypoxic and therefore non-proliferating clonogenic cells. Taken together with previous studies on poorly differentiated FaDu and moderately well differentiated GL hSCC, the results are consistent with considerable variability in the magnitude and kinetics of repopulation in different experimental squamous cell carcinomas, and with a relationship between reoxygenation and repopulation during fractionated irradiation. The differentiation status of hSCC growing in nude mice does not to appear to correlate with the proliferative capacity of clonogenic tumour cells during treatment. The results do not support the hypothesis gained from clinical data of higher repopulation in well-differentiated tumours.     

Late radiation response of kidney assayed by tubule-cell survival

An assay for the survival of renal tubule cells was developed using mice. It is analogous to other in-situ clonogenic cell survival assays. One kidney was irradiated using a 137Cs irradiator and removed 60-68 weeks later for histological examination. In unirradiated animals there were about 370 tubules in contact with the capsule in a coronal cross section at the middle of the kidney. After irradiation, extensive tubular damage was the dominant lesion. The number of epithelialized tubules in contact with the capsule showed a dose-dependent logarithmic decline. The dose-survival relationship for the clonogenic cells responsible for the regeneration of tubule epithelium was described by a D0 value of 1.5 Gy over the dose range 11-16 Gy. This radiosensitivity resembles that of stem cells in acutely responding tissues. The lack of histological evidence of damage to the arterial vasculature at the time the tubules are initially denuded of epithelium, and the similarity of renal tubule cell radiosensitivity to that of other mammalian cells, support the hypothesis that "late" radiation injury results primarily from depletion of parenchymal cells, not indirectly from injury to blood vessels, as has been the prevailing belief.     

Effect of bcl-2 deficiency on the radiation response of clonogenic cells in small and large intestine, bone marrow and testis

PURPOSE: Overexpression of bcl-2 protects against radiation induced apoptosis in lymphohaematopoietic cell types in vivo, whilst bcl-2 deficiency radiosensitizes murine T-lymphocytes in vitro. However, there are few data regarding the influence of bcl-2 deficiency on the radiosensitivity of non-lymphoid cell types. The purpose of this study was to investigate the role of bcl-2 in the clonogenic radiation response of intestinal crypts, bone marrow progenitor cells and testicular stem cells. METHOD: Survival curves were obtained for each cell type from bcl-2 null (-/-), heterozygote (+/-) and wild type (+/+) mice. Crypt survival in the small and large intestine was assessed using the crypt microcolony assay. Committed haemopoietic progenitors were assayed using in vitro colony-forming cell (CFC) assays and survival of clonogenic spermatogonia was assessed by scoring regenerative tubules at 35 days post-irradiation. RESULTS: There was no difference in small intestine crypt survival between the three genotypes. In the colon, there was a tendency towards lower clonogen survival in the +/- and -/- animals. Haemopoietic in vitro CFC from -/- animals showed lower survival in comparison to +/+ mice, but spermatogonial stem cells were comparatively more radioresistant. CONCLUSIONS: Deficiencies in bcl-2 affect the radiation response of different cell populations in small but different ways. This may be due to variations between cells in their innate capacity for apoptosis, their dependence on different members of the bcl-2 family gene and their cell-cycle status and p53 expression.     

Role of mitochondrial DNA in radiation exposure

PURPOSE: To evaluate the role of mitochondrial DNA (mtDNA) following exposure to ionizing irradiation. MATERIALS AND METHODS: We examined two human osteosarcoma cell lines either lacking mtDNA (143B.rho(0)206; rho0 cells) or having normal mtDNA (143B.TK-; rho+ cells). Cell survival curves were generated by using colony formation and micronucleus assay. The delay in population doubling time after irradiation was evaluated with dye exclusion tests. RESULTS: No significant difference was seen between rho+ and rho0 cell lines in colony formation assay. In micronucleus assay, rho0 cells showed a significantly lower rate of micronucleus formation. The ratios of binucleated cells with micronuclei were 0.49 for rho+ cells and 0.25 for rho0 cells (p=0.005). In the dye exclusion test, rho0 cells revealed a delay of about 1.6 times in population doubling time compared with the control after 5 Gy of irradiation, similar to the 1.7 times of rho+ cells. CONCLUSION: In the human osteosarcoma cell line 143B.TK-, mtDNA does not influence clonogenic survival and delay of population doubling time after irradiation. However, the difference in micronucleus formation shows that mtDNA influences DNA damage after radiation exposure.     

Effect of bcl-2 deficiency on the radiation response of clonogenic cells in small and large intestine,bone marrow and testis

Purpose : Overexpression of bcl -2 protects against radiation induced apoptosis in lymphohaematopoietic cell types in vivo, whilst bcl -2 deficiency radiosensitizes murine T-lymphocytes in vitro. However, there are few data regarding the influence of bcl-2 deficiency on the radiosensitivity of non-lymphoid cell types. The purpose of this study was to investigate the role of bcl-2 in the clonogenic radiation response of intestinal crypts, bone marrow progenitor cells and testicular stem cells. Method : Survival curves were obtained for each cell type from bcl -2 null (-/-), heterozygote (+/-) and wild type (+/+) mice. Crypt survival in the small and large intestine was assessed using the crypt microcolony assay. Committed haemopoietic progenitors were assayed using in vitro colony-forming cell (CFC) assays and survival of clonogenic spermatogonia was assessed by scoring regenerative tubules at 35 days post-irradiation. Results : There was no difference in small intestine crypt survival between the three genotypes. In the colon, there was a tendency towards lower clonogen survival in the +/- and -/- animals. Haemopoietic in vitro CFC from -/- animals showed lower survival in comparison to +/+ mice, but spermatogonial stem cells were comparatively more radioresistant. Conclusions : Deficiencies in bcl -2 a ffect the radiation response of different cell populations in small but different ways. This may be due to variations between cells in their innate capacity for apoptosis, their dependence on different members of the bcl -2 family gene and their cell-cycle status and p53 expression.     

Impact of preirradiation of the tumour bed on cell production rate and cell loss of human FaDu squamous cell carcinoma growing in nude mice.

PURPOSE: To explore whether the tumour bed effect (TBE) in FaDu squamous cell carcinoma growing in nude mice is caused by a reduced tumour cell production rate and/or by increased tumour cell loss. MATERIALS AND METHODS: Human FaDu tumours were studied in NMRI nude mice. The volume doubling time (VDT) between 100 and 400 mm3 was determined for tumours in unirradiated subcutaneous (sc) tissues (group 1), tumours in sc tissues preirradiated with 12.5 Gy (group 2), tumours irradiated in situ with 12.5 Gy (group 3), and tumours from group 3 re-transplanted into unirradiated sc tissues (group 4). Labelling index (LI), potential doubling time (Tpot), relative necrotic area and apoptotic index (AI) were evaluated in tumours from groups 1 and 2. RESULTS: The median VDT were 2.6 days (95% CI 2-4) in group 1 and 7.0 days (4-15) in group 2 (p<0.001). The VDT were not significantly different between groups 2 and 3, and group 1 and 4. In groups 1 and 2, the Tpot values (3.1 +/- 0.6 days (SD) versus 2.9 +/- 0.5 days) and the LI were identical (10 +/- 1.5%). The median relative necrotic area was significantly larger in group 2 (37% [23-42]) compared with group (6% [0.3-27]). The apoptotic index was low (0.2%) and did not differ between groups 1 and 2. CONCLUSIONS: The results indicate that the TBE in FaDu squamous cell carcinoma is not caused by a reduced cell production rate in the viable tumour compartment. Rather, the TBE reflects a decreased viable tumour cell compartment due to increased cell loss. Necrosis appears to be the major component of the tumour bed induced cell loss in FaDu tumours, whereas apoptosis has no impact on the TBE in this model.     

Impact of increased cell loss on the repopulation rate during fractionated irradiation in human FaDu squamous cell carcinoma growing in nude mice

Purpose: To determine the impact of increased necrotic cell loss on the repopulation rate of clonogenic cells during fractionated irradiation in human FaDu squamous cell carcinoma in nude mice.

Materials and methods: FaDu tumours were transplanted into pre‐irradiated subcutaneous tissues. This manoeuvre has previously been shown to result in a clear‐cut tumour bed effect, i.e. tumours grow at a slower rate compared with control tumours. This tumour bed effect was caused by an increased necrotic cell loss with a constant cell production rate. After increasing numbers of 3‐Gy fractions (time intervals 24 or 48?h), graded top‐up doses were given to determine the dose required to control 50% of the tumours (TCD₅₀). All irradiations were given under clamp hypoxia.

Results: With increasing numbers of daily fractions, the top‐up TCD₅₀ decreased from 37.9?Gy (95% CI: 31; 45) after single dose irradiation to 14.1?Gy (8; 20) after irradiation with 15 fractions in 15 days. Irradiation with 18 daily 3‐Gy fractions controlled more than 50% of the tumours without a top‐up dose. After irradiation with six fractions every second day, the top‐up TCD₅₀ decreased to 26.9?Gy (22; 32). No further decrease of the TCD₅₀ was observed after 12 and 18 irradiations every second day. Assuming a constant increase of TCD₅₀ with time, the calculated doubling time of the clonogenic tumour cells (T_clon) was 7.8 days (4.4; 11.3). The T_clon calculated for FaDu tumours growing in pre‐irradiated tissues was significantly longer (p=0.0004) than the T_clon of 5.1 days (3.7; 6.5) determined under the same assumptions in a previous study for FaDu tumours growing in normal subcutaneous tissues.

Conclusions: Increased necrotic cell loss by pre‐irradiation of the tumour bed resulted in longer clonogen doubling times during fractionated radiotherapy of human FaDu squamous cell carcinoma. This implies that a decreased necrotic cell loss might be the link between reoxygenation and repopulation demonstrated previously in the same tumour model.     

Zeitfaktor und Tumorrepopulierung während einer fraktionierten Radiotherapie

BACKGROUND: A series of experiments were performed to determine the local tumour control of two human squamous cell carcinoma lines in nude mice. An accelerated-fractionated radiation therapy regime is compared to a conventional-fractionated therapy regime. MATERIAL AND METHODS: KB is a well established human nasopharyngeal squamous cell carcinoma line (ATCC CCL 17). In nude mice KB grows as an low differentiated carcinoma. PEC MB is an undifferentiated squamous cell carcinoma of the maxillary sinus, which was successfully established in nude mice by our group 1993. Both tumors were serially passaged in nude mice. Local irradiation was given without anaesthesia under ambient conditions to air breathing animals using 18 MeV electrons of an linear accelerator (Mevatron 77, Siemens, Munich). Each dose level group consists of six to eight animals. The radiation treatments were given in ten equals fractions using graded dose levels of 2, 3, 4.5, 6 and 8 Gy. The interfraction time interval was 6 hours in the accelerated-fractionated group and 24 hours in the conventional-fractionated group (Figure 1). In the conventional-fractionated group a therapy break was given after 5 fractions for 72 h. The endpoint of the experiments was the dose, which was necessary to control 50% of the tumors (TCD(50)). The TCD(50) values were calculated after 60 days (Tables 1a and 1b). RESULTS: The experiments show, that with increasing overall treatment time of 8 3/4 days using the same number of fractions under ambient conditions the tumor control dose of the tumor KB increases from 36.3 Gy (95% CI 30.9...42.7) to 44.3 Gy (38.3...51.2) (Figure 2a). For the tumor PEC MB the tumor control dose increases from 39.5 Gy (33.4...46.7) to 45.5 Gy (37.0...56.0 (Figure 2b). CONCLUSION: This observed increase of the dose necessary to control the squamous cell carcinoma KB and PEC MB can be caused by repopulation of clonogenic tumors cells, however, other mechanism such as an increasing fraction of hypoxic tumor cells can not be ruled out.