Article

Reproductive Survival of Explanted Human Tumor Cells after Exposure to Nitrogen Mustard or X Irradiation; Differences in Response with Subsequent Subculture in Vitro

Authors:
If you want to read the PDF, try requesting it from the authors.

Abstract

Curves for the survival of reproductive capacity of explanted human tumor cells, following exposure to the alkylating agent nitrogen mustard (mustine hydrochloride) or 250-kVp x rays, were obtained as soon as a satisfactory plating efficiency, i.e., greater than or approximately equal to 10 percent, was obtained from the tumor cells in vitro (usually within 2-10 weeks of explanation). It was found that all six tumor explants tested became more sensitive to the action of nitrogen mustard on serial subculture, whereas the response of four explants which were X-irradiated was invariant with further subculturing. Furthermore, all but one explant yielded survival curves which were extremely similar, with D/sub q/ values circa 440-610 rad. One line, from a seminoma, however, had a D/sub q/ of 150 rad. These radiosensitive seminoma cells were, however, the most resistant to the action of nitrogen mustard. The increase in sensitivity to nitrogen mustard with serial subculture in vitro was not associated with any change in the proliferative rate of the cells, although it may be associated with an increase in the efficiency of transport.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

Article
By predicting treatment outcome to radiotherapy from in vitro radiobiological parameters, not only individual patient treatments can be tailored, but also new promising treatment protocols can be tried in patients in whom unfavorable outcome is predicted. In this respect, choosing the right parameter can be very important. Unlike D0 and N which provide information of the distal part of the survival curve, mean inactivation dose (Δ) estimates overall radiosensitivity. However, the parameters reflecting the response at the clinically relevant low-dose region are neglected in the literature. In a literature survey of 98 papers in which survival curves or D0/N were used, only in 2 Δ was used. In 21 papers the D0/N values were important in drawing conclusions. By calculating Δ in 3 of these 21 papers, we show that the conclusion drawn may be altered with the use of Δ. The importance of ‘low-dose-region-parameters’ is reviewed.
Article
A statistical analysis has been performed on a set of 59 published survival curves of human cell lines. Six fibroblast cell lines derived from patients free of genetic disorders and 36 tumor cell lines were used in this study. Using the linear quadratic (L-Q) model, which provides an overall adequate fitting, especially in the low dose range, we show that great variations in radiosensitivity exist among cell lines. In the low dose range (≅2 Gy), these variations cannot be explained on the mere basis of technical factors. Cell type is described as an intrinsic radiosensitivity factor, as variations of mean radiosensitivity among cell types are statistically significant at low doses. A correlation is found between the 95% tumor control dose and the mean surviving fraction at 2 Gy for a given cell type. Higher radiosensitivity is accompanied by lower tumor control dose (TCD 95 % ). This correlation suggests that the moderate radiocurability of certain tumors can be partially explained by the intrinsic radiosensitivity of relevant tumor cells and in particular by a high surviving fraction at 2 Gy.
Article
The role of radiation therapy is essential in the treatment of endometrial carcinoma. The present knowledge of the radiation sensitivity of endometrial carcinoma is mostly empirical and based on clinical trials. To study the inherent radiation sensitivity of endometrial carcinoma, we tested two long-established cell lines (RL95-2, KLE) and four new, low-passage cell lines (UM-EC-1, UM-EC-2, UM-EC-3, UT-EC-1) by using a 96-well plate clonogenic assay. This method has proved to be suitable for clonogenic studies of both squamous cell carcinomas (SCC) and adenocarcinomas in our recent works. Plating efficiencies of the cell lines tested varied from 0.005 to 0.45. Cells were irradiated in suspension with a 4-MeV linear accelerator at a dose rate of 2.0 Gy/min. Survival data were fitted by the linear quadratic function F = exp (-(alpha D + beta D2)). Radiation sensitivity was expressed as the area under the curve (AUC), equivalent to the mean inactivation dose. UM-EC-1 and UT-EC-1 were the most radiation-resistant cell lines tested (AUC greater than 1.6 Gy), while UM-EC-3 was the most sensitive (AUC = 1.0 Gy). The difference in radiation sensitivity between these cell lines was statistically significant (P less than 0.001). As a group, endometrial carcinoma cell lines were clearly more radiosensitive than most SCC lines of head and neck and vulva earlier tested by us. However, our results showed also great variance in the inherent radiation sensitivity between individual cell lines derived from endometrial carcinomas.
Article
By predicting treatment outcome to radiotherapy from in vitro radiobiological parameters, not only individual patient treatments can be tailored, but also new promising treatment protocols can be tried in patients in whom unfavorable outcome is predicted. In this respect, choosing the right parameter can be very important. Unlike D0 and N which provide information of the distal part of the survival curve, mean inactivation dose (D) estimates overall radiosensitivity. However, the parameters reflecting the response at the clinically relevant low-dose region are neglected in the literature. In a literature survey of 98 papers in which survival curves or D0/N were used, only in 2D was used. In 21 papers the D0/N values were important in drawing conclusions. By calculating D in 3 of these 21 papers, we show that the conclusion drawn may be altered with the use of D. The importance of 'low-dose-region-parameters' is reviewed.
Article
Tumor cells (SGC-7901) obtained from a human gastric adenocarcinoma have been examined with regard to their intrinsic sensitivity to gamma-irradiation and as to how this intrinsic sensitivity might be altered by growth in a synthesized agent AT-1727 (N4-morpholino-methyl-3,5-dioxopiperazynyl-1,2 ethanl). Clonogenic survival was measured via colony formation assays and fitted to single-hit-multitarget formula. Exponentially growing cells exhibited the mean of survival parameters: D0 = 0.86 + 0.04 Gy; n = 7.03 + 2.3; Dq = 1.63 + 0.23 Gy and SF2 (surviving fraction of conventional daily clinical dose of 2 Gy) = 45-50%. Split-dose survival assays and delayed plating methods were used to exploit the capacity of sublethal damage repair of this cell line studied demonstrating the reappearance of initial shoulder on the survival curve and an increased survival. Alteration of radiosensitivity of SGC cells by AT-1727 was shown when cultures were incubated in a medium with the drug for a 6 hr interval between split dose irradiation, indicating the inhibition of sublethal damage repair. The radioresistance of hypoxic SGC cells was decreased when pre-treatment with AT-1727 was given 2 hr before irradiation, and the decrease of surviving fraction was drug-dose dependent. A maximum enhanced effect was obtained when 0.15 mM of AT-1727 was used, reaching an ER of 1.24. Inhibition of sublethal damage repair and action as a hypoxic radiosensitizer were considered to be two parts of the mechanism of AT-1727 in modification of radiation effects.
In vitro effects of radiation were studied in two permanent cell lines (AGS and SII) from two patients with adenocarcinoma of the stomach and three permanent sublines from each cell line. Radiation survival parameters for AGS and SII parent cell lines and sublines were determined after in vitro irradiation of their cells with 0.5 to 10 Gy of 60Co gamma rays. The AGS and SII cell lines had different growth properties, DNA contents and radiation survival curves. Surviving fractions of SII parent cells (76 chromosomes) after 2.0 and 10 Gy were 1.22 and 17.8 times greater, respectively, than values for AGS parent cells (47 chromosomes). Sensitivities (D0) were 1.08 and 1.45 Gy for AGS and SII parent lines, respectively. The D0 values for AGS parent cells and sublines were similar (1.01 to 1.08 Gy), but SII parent cells and sublines had D0 values of 1.45, 1.36, 1.37 and 1.12 Gy (for SII-A). Also, the SII parent cells had survival fractions after 2.0 and 10 Gy that were 1.3 and 11.3 times greater, respectively, than values for the SII-A cells. These data show differences in radiation responses among stomach cancer cell lines and sublines that may relate to DNA content, but there was no consistent correlation between radiation response and a particular cell characteristic.
Article
Eleven human lung tumour lines have been established in xenograft or tissue culture, and the responses to acute irradiation of the 10 lines which cloned in soft agar were assayed. In vitro radiosensitivity was evaluated using the multitarget and linear quadratic models of cell survival and the surviving fraction at 2 Gy. Significant differences in the response of the different cell types were found, the large-cell phenotype exhibiting radioresistance, and small-cell carcinomas and adenocarcinomas being radiosensitive. No differences in the capacity of the different tumour types to repair radiation damage were demonstrated. In vivo and spheroid response was modified by the effects of hypoxia and cell-contact phenomena. The results suggested that hyperfractionation would be useful in the clinical management of adenocarcinoma and small-cell carcinoma.
Article
In this paper we review the current data on the role of potentially lethal damage (PLD) recovery in human tumour cell lines, both in vitro and in vivo. In the case of cell lines studied in vitro, the mean recovery ratios found were higher for cells derived from tumours of low curability (glioblastoma, hypernephroma, osteosarcoma, melanoma) than for cells derived from tumours of high curability (breast carcinoma, neuroblastoma). Experiments were performed in vivo only with tumours of low and intermediate curability (melanoma, adenocarcinoma of the colon, pancreatic tumour). Although fragmentary and obtained only with established cell lines, these results argue in favour of the occurrence of PLD repair in human tumour, the amplitude of this repair being, in certain cases, sufficient to explain the incurability of a tumour by radiation therapy.
Article
Clonally-derived cells from ten human malignant tumors considered radiocurable (breast, neuroblastoma, medulloblastoma) or non-radiocurable (osteosarcoma, hypernephroma, glioblastoma, melanoma) were studied in cell culture and their in vitro x-ray survival curve parameters determined (, Do). There were no significant differences among the tumor cell lines suggesting that survival parameters in vitro do not explain differences in clinical radiocurability. Preliminary investigation with density inhibited human tumor cells indicate that such an approach may yield information regarding inherent cellular differences in radiocurability.
Article
Curves for the survival of reproductive capacity in vitro were obtained for freshly explanted human tumor cells and the standard human tumor cells line HeLa S-3/sub oxf/ following x irradiation. The results suggest that preirradiated feeder cells have a more significant effect at doses of about 700 to 900 rad (in terms of increased survival of an initially viable irradiated test cell population) than effective feeder cells produced in the test cell population. A direct effect of using irradiated feeder cells is an increase in the size of the shoulder as measured by D/sub q/ and an increase in the gradient of the high-dose region of the survival curve.
Article
We have studied the repair of X-ray-induced, potentially lethal damage (PLD) in 9 human tumour lines derived from tumours of varying radiocurability. Cells derived from 3 tumours considered non-radiocurable (1 osteosarcoma, 2 melanoma) repaired significantly more X-ray PLD than cells from 3 tumours considered radiocurable (2 breast, 1 neuroblastoma). The remaining tumour lines were intermediate in their ability for repair, and included cells from another osteosarcoma, a hypernephroma and a glioblastoma. We conclude that the repair of X-ray PLD may be an important cellular determinant of clinical radiocurability.
Article
The radiosensitivity of two human cell lines, Ma 11 and Be 11, each arising from a human melanoma, was studied. The tumors were transplanted into nude mice and irradiated in situ, and the colony-forming ability was tested in vitro. The Dâ values obtained under these conditions were 4.22 and 5.85 Gy for Ma 11 and Be 11, respectively. The surviving fraction of irradiated cells increased when tumor excision was delayed for 6 hr. The Be 11 line contained 40% hypoxic cells; the corresponding figure for Ma 11 was 62%. These figures like that previously determined for another malanoma (85%), are among the highest reported in the literature. For solid tumors, only 10% of tumors have a hypoxic cell fraction greater than 40%; this high fraction may partially explain the radioresistance of human melanomas.
Article
The intrinsic radiosensitivity of human cell lines (five tumor and one nontransformed fibroblastic) was studied in vitro. The survival curves were fitted by the single-hit multitarget, the two-hit multitarget, the single-hit multitarget with initial slope, and the quadratic models. The accuracy of the experimental results permitted evaluation of the various fittings. Both a statistical test (comparison of variances left unexplained by the four models) and a biological consideration (check for independence of the fitted parameters vis-a-vis the portion of the survival curve in question) were carried out. The quadratic model came out best with each of them. It described the low-dose effects satisfactorily, revealing a single-hit lethal component. This finding and the fact that the six survival curves displayed a continuous curvature ruled out the adoption of the target models as well as the widely used linear regression. As calculated by the quadratic model, the parameters of the six cell lines lead to the following conclusions: (a) the intrinsic radiosensitivity varies greatly among the different cell lines; (b) the interpretation of the fibroblast survival curve is not basically different from that of the tumor cell lines; and (c) the radiosensitivity of these human cell lines is comparable to that of other mammalian cell lines.
Article
To determine whether some of the deviations from the simple linear-quadratic (LQ) theory in the radiation dose survival responses of asynchronous cultures of human tumour cell lines are caused by the presence of cell-age specific subpopulations which all individually follow LQ theory, but have different radiosensitivities. Human tumour cells were synchronized by mitotic selection and their survival dose responses were measured at doses from 0.05 Gy to 12 Gy, using a high-precision survival assay. These responses were compared with a kinetic model of radiation survival in synchronized cells, which assumed that age-specific populations individually obeyed the LQ theory. The cell lines used included HT-29 and A549, which have typical dose responses, and U1, which is somewhat atypical. In two of the three cell lines, A549 and HT-29, observed deviations from the LQ model were consistent with those expected from cell-age heterogeneity. In the third cell line, U1, survival responses could not be described by the LQ theory, even when cell-age heterogeneity was considered. The LQ model provided an adequate description of cell survival for two of three tumour cell lines in this study when cell-age related heterogeneity in survival responses was accounted for. However, some alternative survival models (such as the repair saturation model) provided better characterizations of the survival response of the third cell line and, in fact, gave good descriptions of survival for all three cell lines.
Article
Full-text available
Ninety-seven fresh explants of human tumours have been cultured in vitro in an attempt to predict their sensitivity to subsequent cytotoxic chemotherapy. Only 3/26 solid ovarian tumours were cultured successfully although 12 of the 23 which failed to grow proved later to have benign histology. Of 10 solid tumours from other sites, only 2/4 renal tumours and one melanoma were successfully grown and tested in vitro. A higher success rate was achieved in culturing carcinoma cells of ovarian (10/22) and breast (10/22) origin from ascitic and pleural fluids. Using increase in cell number after 7 days' growth in vitro as the biological end-point, concentrations of cytotoxic drugs which are achievable in patients produced significant effects on some tumour explants. Detailed studies of serial subcultures in vitro from an ovarian tumour showed that large changes in chemosensitivity occur within about 2 passages, in vitro, so that sensitivity testing can only be carried out using fresh explants or their first subcultures if any possible correlation between in vitro cytotoxicity and in situ response is to be studied. Clinical effectiveness and in vitro chemosensitivity are compared for a limited number of patients with ovarian and breast carcinomata for whom follow-up information was available; no useful correlation was found.
Article
The skin reaction of the irradiated mouse leg has been used to determine the ‘sparing’ effect of dose-fractionation on a normal mammalian tissue. The total dose in two fractions (D2) required to produce the same effect as a single fraction (D1) was determined for intervals of 2 to 42 hours. A rapid increase of D2 – D1 up to 4–5 hours was observed. Irradiations were carried out in oxygen, air and nitrogen and the values obtained for (D2 – D1) at 24 hours in air and oxygen were in the range 300–880 rads (15 mev electrons). These agree well with the findings previously reported on pig and human skin. Hypoxia has been demonstrated to affect the response of the mouse skin irradiated when the animal was breathing air or oxygen at normal atmospheric pressure.
Article
A SIMILARITY of the mode of action of some chemical and physical agents (nitrogen mustards, ultra-violet and X-rays) has been proposed, on the basis of the similarity of many biological end-effects (pharmacological and toxicological properties, mutagenic effects, growth inhibition). Although several authors have suggested that this may be the result of reactions initiated by fundamentally different primary events, of significance to the interpretation of a common mode of action of both alkylating agents and ionizing radiations would be a description of the cross-resistance phenomenon in strains of Escherichia coli 1,2.
Article
THERE have been several attempts to define conditions necessary for the fixation of the transformed state in mammalian cells in vitro. The relationship between fixation and proliferation after treatment has been studied in cell cultures exposed to methylcholanthrene1, X irradiation2,3 and SV40 virus4. Cells were exposed to the carcinogen while in the density-inhibited plateau phase of growth, and subsequently subcultured at low cell density so that they could resume cell division. Transformation frequency was always reduced by delaying subculture for 24 h or longer, suggesting that cells must divide soon after exposure to a carcinogen in order to fix the transformed state. No data were reported for subculture less than 24 h after treatment.
Fractionated doses of 240 kV X-rays were given to one hind leg of mice, and skin reactions were recorded up to 5 weeks after the last dose. The following schedules of irradiation were used: 15 equal fractions in 18 days (15F/18d), 9F/18d, 9F/10d, 5F/9d, 5F/4d, 3F/4d, 2F/2d and single doses. The dose required to produce a given average skin reaction over the period 10 to 32 days, or a corresponding period after multifraction doses, increased steadily with time and fraction number; except for the 9F/10d schedule which produced reactions significantly less than would be expected from any simple sequence. The results are discussed in comparison with other experiments on skin. After allowing for concurrent proliferation, a dose–response curve was derived which had a Dq of 550 rads and a straight portion for doses above 1200 rads. The ratio of initial to final slopes was approximately 0·4 and this indicated the ratio of irreparable to reparable X-ray injury in the skin.
Article
SUMMARY Sensitivity of L5178Y lymphoblasts to the alkylating agent nitrogen mustard (HN2) was found to be a function of the proliferative state of the cells. Dose-survivalcurves showed that exponential or log-phasecellswere 2.16-fold more sensitive to the drug than were stationary-phase cells. Carrier-mediated transport of '"C-labeled HN2, hydrolyzed HN2, and the natural substrate choline was also a function of the proliferative state of the cells. Transport was more efficient in log-phase cells as manifested by a higher binding affinity between carrier and each of the three substrates; in addition transport capacity for hydrolyzed HN2 and choline was significantly greater in log-phase cells than resting cells. The greater sensitivity of log-phasecells to HN2 to a large extent can be accounted for by a more efficient transport mechanism. The activity of the transport system for HN2 may be dependent upon proliferative rate, transport being more active in rapidly dividingcells.
Article
Removal of clone A Chinese hamster and HeLa S-3oxf. cells from monolayer cultures into suspension, followed by replating, produces a partial synchronization of the population in both cell lines. This is reflected by changes in the sensitivity of the cells to the killing effects of X-rays. In Chinese hamster cells, trypsinization was shown to produce a transient suppression of the incorporation of 3H-thymidine into DNA. In the early hours following trypsinization the perturbations in X-ray response are consequences of the trypsinization procedure and are qualitatively similar in both cell lines. Later changes in response reflect the increasing importance of the most resistant fraction of the cell population in determining the survival curves.
Mouse L cells growing in vitro in log phase were irradiated with either ; Co⁶° gamma rays or 220-kvp x rays, and the inhibition of hoth cellular ; multiplication (growth) and colony formation were studied. Growth inhibition is ; greatly dependent upon the length of time after irradiation that it is measured; ; however, the suppression of colony formation is relatively independent of the ; time of scoring. Growth ihhibition measured at 3 to 4 days after irradiation ; showed less radiation effect than did the suppression of colony formation; ; whereas, growth inhibition measured at 9 to 10 days after irradiation showed a ; greater radiation effect, even after correction for the initial mitotic lag. The ; greater radiation effect exhibited by growth inhibition was present because the ; colonies that developed from cells surviving irradiation grew at a reduced rate. ; This decrease in the proliferation rate may be attributed either to an increase ; in the time required for the irradiated cells to complete their cell cycle or to ; some of the descendants of the surviving cells failing to divide. (auth);
Article
The dose required to kill mice three to four days after whole body x irradiation is compared after single and after fractionated doses. It is assumed that death at three to four days after irradiation is due to the reduction of the crypt cells of the small intestine to a constant, though unknown, survival level. The difference in the dose required to produce this effect after single or fractionated doses gives a measure of the size of the shoulder on a Type C survival curve. The results suggest that the shoulder of the survival curve for the crypt cells of Leiberkuhn is large, with D/sub Q/ more than 400 r and n more than 28. (auth)
Article
Calculations of total doses for different numbers of fractions are presented, for two special assumptions. One is based on published radiotherapy results, i.e. on normal tissue reactions and cure of squamous-cell carcindoma, together with experiments on the skin of pigs and mice. The other is based on a cell survival curve model with extrapolation number = 2·8 and D0 = 140 rads. Neither assumption allows fully for cell repopulation, and the significance of this is discussed. The limitations of the calculations as a guide to radiotherapeutic practice are stressed.