Heterogeneity in surface antigen of cell lines and clones from a murine carcinoma

Cell lines of the murine Lewis lung tumor (3LL) were established from primary tumor site (PT) and from lung metastatic foci (Met). Expression of histocompatibility antigens was assessed by immunofluorescence with an anti-H-2b antibody and by tumorigenicity of the cells in different strains of mice. Analysis for tumor-associated transplantation antigens (TATA) were performed by challenging mice with living tumor cells after pretreatment with irradiated cells or by in vivo tumor neutralization assays with different tumor cells as target cells and T lymphocytes from immunized animals as effector cells. The 3LL tumor was found to be heterogeneous in respect to tumor growth rate in vivo, metastatic capacity in vivo, expression of normal histocompatibility antigens and expression of "TATA". Moreover, monoclonal antibody B1 (H11-115) binding to a Met cell surface antigen, but not to normal cells, was obtained. The antibody bound to 3LL tumor cell lines from primary tumor (PTH, PTV), from a mixture of metastatic foci (MetH, MetV), from different metastatic foci originating from 3 different mice, and to 21 subclones derived from one of the lines established from a metastatic focus. Analysis of the 3LL sublines demonstrated a significant antigenic heterogeneity within each tumor population as well as among the different sublines.     

Heterogeneity in heat sensitivity and development of thermotolerance of cloned cell lines derived from a single human melanoma xenograft

One uncloned and five cloned cell lines were isolated from a single human melanoma xenograft in passage 39 in athymic mice. Cells from passages 7-12 in vitro were heated at 42.5, 43.5 or 44.5 degrees C and the colony forming ability of the cells was assayed in soft agar. The six cell lines showed individual and characteristic responses to heat treatment. The D0 values of the survival curves were in the ranges 76 +/- 5 to 131 +/- 13 min (42.5 degrees C), 12.5 +/- 1.1 to 22.2 +/- 1.9 min (43.5 degrees C) and 9.4 +/- 1.0 to 15.6 +/- 1.5 min (44.5 degrees C). Cells from all lines developed thermotolerance during protracted treatments at 42.5 degrees C. Thermotolerance was also studied by giving the cells a priming treatment of 43.5 degrees C for 90 min and then, after different fractionation intervals at 37 degrees C, second graded treatments at 43.5 degrees C. Thermotolerance ratio (TTR), i.e. the ratio of the slopes of the survival curves for preheated and single-heated cells, was used as a quantitative measure of the thermotolerance. Thermotolerance developed rapidly for all lines, reached a maximum at 12 or 16 h, and then decayed slowly. Maximum TTR varied among the lines from 4.2 +/- 0.5 to 6.0 +/- 0.9, i.e. within a factor of about 1.4. The survival curves and the TTR-curve for the uncloned line were positioned in the midst of those of the cloned lines. A linear correlation between maximum TTR and heat sensitivity was found for the six lines; maximum TTR decreased with increasing D0 value at 43.5 degrees C. Nevertheless, the lines which were most resistant before thermotolerance developed were also most resistant at maximum thermotolerance.     

Cyclopalladated compounds as chemotherapeutic agents: antitumor activity against a murine melanoma cell line

Palladacycle compounds obtained from N, N-dimethyl-1-phenethylamine (dmpa), phenyl-2-pyridinyl-acetylene and 1-phenyl-3-N, N-dimethylamine-propine, respectively, were complexed to 1, 2 ethanebis (diphenylphosphine) (dppe) ligand to synthesize antitumor cyclopalladated complexes that were tested in vitro and in vivo against syngeneic B16F10-Nex2 murine melanoma cells of low immunogenicity implanted subcutaneously in mice. Complexes were not toxic to mice injected 3 times i.p. with as much as 60 microM/animal/week. Of 3 cyclopalladated complexes that were inhibitory in vitro at low concentrations (<1.25 microM), complex 7a was the most active in vivo, delaying tumor growth and prolonging animal survival. In vitro, binucleate complex 7a caused a collapse of respiratory activity with an abrupt decrease of extracellular acidification on short incubation (up to 100 min), followed by DNA degradation after 24 hr. The apoptosis-like reaction to this Pd-complex was not accompanied by increased levels of caspases 1 and 3. Complex 7a bound to a bacterial plasmid DNA, causing late conformational changes after 24 hr. Two other complexes with different C, N-cycles were also apoptotic and 2 binucleated ones were inactive. These results introduce the palladacycle-dppe complexes as promising antitumor drugs with exquisite structural specificities and for action in vivo and in vitro.     

Induction of stress proteins in murine and human melanoma cell cultures

The induction of stress proteins was studied in two human and two murine melanoma cell lines. Exposure for 1 h to heat (42 degrees C), to ethanol (6%), to arsenate (100 microM) and to disulfiram (50 microM) induced the expression of SPs with apparent molecular weights of 100, 86, 70-72 and 24-26 Kd. Quantitation of the single SPs indicated that the basal level as well as the enhanced synthesis following the various stressors were different in each cell line. The induction of the 100 Kd species occurred in only one murine melanoma and not in the others. The 86 and in particular the 70-72 Kd species were the most prominent groups, whereas the 24-26 SPs were induced only following arsenate and disulfiram exposure in the three melanoma cell lines. In one of the murine melanomas, the expression of SPs was markedly reduced compared to the other cell lines. No definite specific patterns of SP expression could be identified in tumors of the same histologic type.     

Heterogeneity of radiation response of a parent human epidermoid carcinoma cell line and four clones

We examined the radiobiological parameters of a parent tumor cell line and four tumor clones of a human skin squamous cell carcinoma. The parent line and all clones have a tumor morphology, aneuploid karyotype, and the ability to passage infinitely in vitro. The parent cell line and three of four clones formed tumors in nude mice. The parent line, SCC-12, has a D0 of 1.59 Gy and an n of 7.5. In the four clones, D0 ranges from 1.31 Gy to 2.66 Gy and n ranges from 2.1 to 22.8. Potentially lethal damage repair (PLDR) following 11 Gy ranges from 1.7 to 13.1, at 24 hours, however PLDR following equitoxic doses of radiation is similar. The mean inactivation dose value (D) in the parent tumor cell line is 3.48 and ranges from 3.31 to 4.17 in the tumor clones. Radiobiological interpretation of heterogeneity may complicate predictive assays for clinical radiotherapy.     

Heterogeneity in induced thermal resistance of rat tumor cell clones

Four 13762NF rat mammary adenocarcinoma clones were examined for their survival response to heating under conditions that induced transient thermal resistance (thermotolerance). Clones MTC and MTF7 were isolated from the subcutaneous locally growing tumor, whereas clones MTLn2 and MTLn3 were derived from spontaneous lung metastases. There was heterogeneity among these clones in thermotolerance induced by either fractionated 45 degrees C or continuous 42 degrees C heating, but the order of sensitivity was not necessarily the same. The clones developed thermal resistance at different rates and to different degrees within the same time intervals. There was heterogeneity between clones isolated from within either the primary site or metastatic lesions. However, clones derived from metastatic foci did not intrinsically acquire more or less thermotolerance to fractionated 45 degrees C or continuous 42 degrees C heating than did clones from the primary tumor. Further, there was no apparent relationship between any phenotypic properties that conferred more or less thermotolerance in vitro and any phenotypic properties that conferred enhanced metastatic success of these same clones by spontaneous (subcutaneous) or experimental (intravenous) routes in vivo. These tumor clones also differ in their karyotype, metastatic potential, cell surface features, sensitivity to x-irradiation and drugs, and ability to repair sublethal radiation damage. These results provide further credence to the concept that inherent heterogeneity within tumors may be as important in therapeutic success as other known modifiers of outcome such as site and treatment heterogeneity.     

A comparison of heat sensitivity, radiosensitivity and PLDR in four human melanoma cell lines.

Comparison of the heat sensitivity and radiosensitivity of four human melanoma cell lines in culture revealed a large variation in sensitivity amongst the four cell lines. Three of the four cell lines had large shoulders on the survival curves when exposed to hyperthermia (44 degrees C or 45 degrees C). These three cell lines also had demonstrable shoulders on the acute radiation dose response curves. The most radiosensitive cell line did not show a shoulder region in the heat or radiation survival curves (HT-144, Dq = 0.2 Gy). Despite this consistency in the presence or absence of shoulder, there was no correlation between heat and radiation sensitivity in the four melanoma cell lines. Furthermore, regardless of radiosensitivity, all four lines studied showed competent repair of potentially lethal damage. The recovery ratios at a surviving fraction of 0.001 ranged from 5.7 to 7.6. All four lines had a similar cell cycle distribution at the time of treatment, hence the variation observed in the response of these four lines to radiation and heat was not due to differences in cell cycle kinetics. Preliminary results of DNA polymerase-alpha and -beta activities do not demonstrate a clear correlation between cellular levels of these two enzymes and radiosensitivity.     

Phenotypic instability of drug sensitivity in a human colon carcinoma cell line

Colon cancer is one of the tumors most refractory to treatment by chemotherapy, and this may be due to inherent phenotypic instability of such tumor cells with respect to the biochemical determinants of drug sensitivity. To test this hypothesis, a clonal human colon carcinoma cell line, clone A, was passaged in culture in the absence of selection conditions or mutagens. During this time, sensitivity to several drugs was examined, and was found to decrease 4-fold during 30 weeks of culture. Five randomly selected subclones, having never been exposed to drug or mutagen, displayed a range of sensitivities to etoposide (50% inhibitory concentrations ranging from 1.5 to 4.9 microM) and to vincristine (9-fold range), but all had the same sensitivity to methotrexate. With time these sensitivities also changed, and subsequent subclones were chosen from the lines with highest and lowest drug sensitivity. Again a wide range of phenotypes was observed. Sensitivity to vincristine ranged 14-fold and to doxorubicin 3-fold. Several biochemical determinants of drug sensitivity had a broad range of expression between cell lines. Cellular accumulation of [3H]vincristine, as well as expression of multidrug resistance protein P170 and glutathione transferase activity all varied significantly between subclonal lines. This suggests that some human colon tumors may be phenotypically unstable with respect to drug sensitivity, and this could contribute to clinical resistance to chemotherapeutic compounds.     

人子宫内膜癌体外化学药物敏感性的实验研究

目的 利用人子宫内膜癌细胞株(HECCL-1)筛选对人子宫内膜癌敏感的化学药物,并探讨其作用机制。方法 采用四甲基偶氮唑蓝(MTY)法体外药敏实验检测HECCL-1细胞对18种不同浓度的化学药物的敏感性。利用流式细胞仪(FCM)进行细胞周期分析,并检测细胞凋亡率及用药前后多药耐药基因MDR1的表达。结果 MTY法结果显示,部分化学药物可显著抑制HECCL-1细胞的增殖,且呈剂量依赖效应,敏感性药物依次为阿霉素、奥铂、卡铂、顺铂、泰素、表阿霉素、放线菌素D、米托葸醌和氟脲嘧啶。FCM检测结果显示,化学药物作用后G0期、G1期细胞显著减少,S期、G2期、M期细胞显著增加。在血浆峰值浓度(PPC)下,11种化学药物作用于HECCL-1细胞后出现细胞凋亡图像。表阿霉素、氟脲嘧啶、羟基喜树碱、米托葸醌4种药物作用后诱导出MDR1阳性表达。结论 HECCL-1是一种化学药物较敏感的细胞株,多种化学药物能够显著抑制HECCL-1细胞的增殖活性,改变其细胞周期分布;诱导细胞凋亡是化学药物作用于该细胞的重要机制;获得性耐药是继续用药失败的主要原因。     

Swainsonine protects both murine and human haematopoietic systems from chemotherapeutic toxicity.

The haematopoietic system is sensitive to cytotoxic damage and is often the site of dose-limiting toxicity. We previously reported that swainsonine, an inhibitor of protein glycosylation, reduced the bone marrow toxicity resulting from a single dose of anticancer drugs in otherwise healthy mice. However, more important questions are (1) can swainsonine protect tumour-bearing mice without interfering with the anti-tumour effects of the drugs, and (2) can swainsonine stimulate haematopoietic activity of human, as well as murine, bone marrow. We demonstrate here that swainsonine protects C57BL/6 mice bearing melanoma-derived tumours from cyclophosphamide-induced toxicity without interfering with the drug's ability to inhibit tumour growth. Similar results were obtained in vivo with 3'-azido-3'-deoxythymidine (AZT), a myelosuppressive agent often used in therapy for acquired immune deficiency syndrome. Swainsonine increased both total bone marrow cellularity and the number of circulating white blood cells in mice treated with doses of AZT that typically lead to severe myelosuppression. Swainsonine also increased the number of erythroid and myeloid colony forming cells (CFCs) in short-term cultures of murine bone marrow, restoring the number of progenitor cells to the control level in the presence of AZT doses that reduced CFCs by 80%. With respect to the sensitivity of human haematopoietic cells to swainsonine, we show that swainsonine protected human myeloid progenitor cells from AZT toxicity in vitro. These results suggest that swainsonine may be useful as an adjuvant in several types of human chemotherapy.     

胰岛素对人乳腺癌细胞株化疗增效作用的体外研究

目的：探讨用胰岛素提高肿瘤细胞生长代谢水平对化疗药物敏感性的影响。方法：以体外培养的人乳腺癌细胞株（MBA-MD-543）为靶细胞,以胰岛素作为代谢促进剂,运用四氮唑盐、直接细胞计数等方法观察化疗药物诺维本与胰岛素对MBA-MD-543细胞活力及细胞代谢,细胞总数等的影响。结果：胰岛素（0．5～16mU／mL,8～18h）使MBA—MD-543的细胞活力及细胞代谢增加,胰岛素（7．5mU／mL）可以增加诺维本的细胞毒作用。结论：在体外,胰岛素可诱导人乳腺癌细胞株MBA-MD-543增殖,胰岛素对诺维本有一定的化疗增效作用,其用药时机和浓度的选择是实现增效的关键。“提高癌细胞的生长代谢水平,继之给予化疗,可提高化疗药物细胞毒作用”是可行的。     

Retention of cellular radiation sensitivity in cell and xenograft lines established from human melanoma surgical specimens.

Six human melanoma xenograft lines have been established in athymic mice from metastatic lesions in six different patients. Permanent cell lines in monolayer culture have been established from four of the xenograft lines. The cellular radiation sensitivity of the donor patients' tumors, the xenograft lines, and the cell lines were measured in vitro. The Courtenay soft agar colony assay was used for the donor patients' tumors, and a conventional plastic surface colony assay was used for the cell lines, whereas both assays were used for the xenograft lines. The cell survival data were analyzed using the multitarget single-hit as well as the linear-quadratic model. The donor patients' tumors differed considerably in cellular radiation sensitivity (the D0 ranged from 0.85 +/- 0.08 to 1.17 +/- 0.09 Gy, the alpha from 0.25 +/- 0.06 to 0.87 +/- 0.14 Gy-1, and the surviving fraction at 2.0 Gy from 0.15 +/- 0.04 to 0.50 +/- 0.06). The xenograft lines showed similar survival curves in soft agar and on the plastic surface, and the survival curves of a xenograft line and the corresponding cell line were not significantly different. These survival curves were not significantly different from those of the donor patients' tumors, regardless of which survival curve parameter was considered, i.e., the cellular radiation sensitivity of the donor patients' tumors was retained in the cell and xenograft lines. Moreover, the cell and xenograft lines have growth properties in vitro and in vivo that render a wide variety of experiments possible. Consequently, they show great promise for future studies of human tumor radiation biology.     

DNA methylation levels in human and murine melanoma cell lines of varying metastatic potential

DNA methylation levels were measured in a series of murine and human melanoma cell lines consisting of matched variants of low and high experimental metastatic capacity. The percentage of cytosine residues modified to 5-methylcytosine ranged between 2.13-3.92% in these lines. Ten cell lines were established in culture from individual lung tumor nodules produced in nude mice by i.v. injection of DX-3 human melanoma cells. Upon reinjection into groups of nude mice the individual lines manifested marked diversity for lung nodule formation (median number of pulmonary tumor nodules ranging from less than 10/group-greater than 100/group). DNA methylation levels in these lines were also heterogeneous (range 1.59 +/- 0.13 (SD)-4.04 +/- 0.15%) but no correlation was detected between methylation status of the genomic DNA and metastatic capacity.     

Biochemical regulation of adenylate cyclase in murine melanoma clones with different metastatic properties

The regulation of adenylate cyclase in murine melanoma tumor cell clones with different metastatic capacities has been studied in intact cells and isolated membrane preparations. Analysis of the responses of intact cells from a number of B16 melanoma clones revealed that treatment with melanocyte-stimulating hormone (MSH) or the diterpene, forskolin, produced significantly greater accumulation of intracellular cyclic adenosine 3',5' monophosphate (cAMP) in strongly metastatic clones than in weakly metastatic tumor cell clones. In contrast, in isolated membranes from the same panel of clones, the extent of activation by forskolin but not by MSH correlated with metastatic capacity. Sodium fluoride and 5'-guanyl-beta-gamma-imidodiphosphate [Gpp(NH)p] also stimulated adenylate cyclase in isolated membranes but the extent of activation did not correlate with the metastatic behavior of the donor cells. A combination of forskolin and Gpp(NH)p proved to be a sensitive prospective indicator for identifying differences in the metastatic capabilities of individual B16 melanoma clones. Adenylate cyclase in membrane preparations from strongly metastatic B16 clones displayed synergistic activation but stimulation of the enzyme from weakly metastatic clones was less than additive. To test the generality of these findings, similar investigations were performed on B16-BL6 melanoma cells, a highly invasive subline of the B16 melanoma, and the K1735, an ultraviolet-light-induced murine melanoma arising in a different mouse strain (C3H). Consistent with their high metastatic potential, clones derived from the B16-BL6 melanoma displayed elevated levels of hormonally-stimulated adenylate cyclase, thereby confirming, for this tumor system, a close association between hormonal responsiveness and metastatic capacity. In contrast, K1735 melanoma cell clones exhibited significant interclonal variation in adenylate cyclase activity and metastatic performance, but no consistent relationship between the two traits was detected. Differences in the regulation and/or the intrinsic catalytic capacity of adenylate cyclase may account, at least in part, for the variation in hormonal responsiveness observed among B16 clones with distinct metastatic properties and suggest that cAMP-dependent molecular processes may be required for the expression of B16 melanoma experimental metastatic potential.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heterogeneity of karyotype and growth potential in simian virus 40-transformed Chinese hamster cell clones.

Five clones of Chinese hamster cells transformed with simian virus 40 (SV40) were isolated from methylcellulose and characterized as to Giemsa-banded karyotype, DNA content, saturation density, agglutination with concanavalin A, and tumorigenicity. Chromosome analysis and DNA content studies at early passage revealed that the genetic complement for all clones was predominantly near tetraploid. All cultures examined contained a proportion of hypertetraploid cells. Nonrandom chromosome changes included at least one broken No 1 chromosone in 80% or more of the cells in each clone, and fewer sex chromosomes than anticipated from the ploidy of the cells. Several abnormal marker chromosomes tended to recur. These changes were more pronounced in the cells cultured from tumors formed by three of the clones. A karyotypically stable stem line was not noted for any of the clones or tumors. The functional significance of the karyotypic heterogeneity was assessed by means of cloning efficiencies both on plastic and in methylcellulose.     

Heterogeneity of chemosensitivity in six clonal cell lines derived from a spontaneous murine astrocytoma and its relationship to genotypic and phenotypic characteristics

Heterogeneity in drug sensitivity must, in part, account for the relative lack of success with single agent chemotherapy for glioblastoma multiforme (GBM). In order to develop in vitro model systems to investigate this, clones derived from the VM spontaneous murine astrocytoma have been characterised with regard to drug sensitivity. Six clonal cell lines have been tested for sensitivity to a panel of cytotoxic drugs using an intermediate duration ³⁵S-methionine uptake assay. These lines have previously been extensively characterised with regard to morphological, antigenic, kinetic, tumourigenic potential in syngeneic animals and chromosomal properties and display considerable heterogeneity. The present study indicates that heterogeneity extends to sensitivity to all classes of cytotoxic drugs. The greatest difference in sensitivity between the clones was seen in response to cell cycle-specific drugs like the Vinca alkaloids (14-fold and 20-fold for vincristine (VCR) and vindesine (VIND) respectively), while the nitrosoureas, CCNU and BCNU displayed a smaller fold difference in sensitivity (4.3 and 3.6-fold difference respectively). All the clones were considerably more resistant to the adriamycin (ADM), cis-platinum (C-PLAT) and the Vinca alkaloids than the parental cell line although the difference in sensitivity between the clones and parental cell line were less marked for the nitrosoureas and procarbazine (PCB). It has also been possible to examine the relationship between drug sensitivity and the phenotypic and genotypic properties of these clonal cell lines. There is a relationship between chromosome number and sensitivity of a wide variety of cytotoxic drugs including the nitrosoureas, Vinca alkaloids, PCB, C-PLAT, BLEO but not ADR or 5-FU. Clones with small numbers of chromosomes were more resistant than clones with gross polyploidy. Similarly, sensitivity to Vinca alkaloids and ADM, but not other classes of drugs, was greatest in cells with numerous cytoplasmic processes and which did not express large amounts of cell surface fibronectin. Preliminary experiments have been conducted on reconstituting clonal mixtures of cells with different sensitivity to Vinca alkaloids and results from these studies indicate that the drug resistance phenotype is dominant, with clonal mixtures of sensitive and resistant cell adopting the sensitivity of the more resistant partner. These cell lines should prove to be useful models for examining the cell biological basis of drug resistance in glioma and may lead to the identification and exploitation of novel cellular targets in new therapies for GBM.     

Effect of transient hypoxia on sensitivity to doxorubicin in human and murine cell lines

Overreplication of DNA associated with gene amplification and drug resistance has been reported to occur after transient hypoxia of rodent cells. Because oxygen levels fluctuate in solid tumors, clinical drug resistance might be stimulated by this mechanism. We have therefore studied the effect of transient hypoxia on sensitivity to doxorubicin in human and murine cell lines. Exposure to hypoxia led to a decreased rate of cell proliferation, and most of the observed changes in sensitivity to doxorubicin were consistent with cell cycle-dependent cytotoxicity of this drug. After transient hypoxia, about 10% of the murine cells (EMT6/Ro and KHT-LP1) contained greater than four times the haploid DNA content (greater than 4C DNA), but only 0%-5% of the human cells (MGH-U1, A549, and Hey) had greater than 4C DNA content. Murine cells that had been exposed to hypoxia and reoxygenation, and which had greater than 4C DNA content, were separated by flow cytometry. For KHT-LP1 cells, but not for EMT6/Ro cells, this subpopulation was found to be more resistant to doxorubicin than the subpopulation with less than 4C DNA content and the aerobic control. When resistant KHT-LP1 clones were expanded in the presence of doxorubicin, six of six clones showed amplification of the P-glycoprotein gene family. The ability and efficiency of hypoxia to induce DNA overreplication, gene amplification, and drug resistance appears to be cell-line dependent.     

Heterogeneity of the induction of HLA-DR expression by human immune interferon on glioma cell lines and their clones

The modulation of HLA-DR and HLA-A, -B, and -C by human recombinant immune interferon (IFN-gamma) was studied on 10 malignant glioma cell lines established in our laboratory, on 8 clones or subclones derived from these lines, and on a fetal astrocyte cell line. Comparative studies were performed with recombinant leukocyte interferon (IFN-alpha). The results not only confirmed the selective activity of IFN-gamma on the modulation of HLA-DR expression, as opposed to that of IFN-alpha, but also demonstrated a marked heterogeneity in the response of glioma cell lines and their clones to the two types of IFN tested. For example, all 3 clones of an inducible cell line could be modulated to express HLA-DR, whereas only 2 of 5 clones derived from a noninducible line were modulated. This heterogeneity did not seem to be due to the absence of the receptor for IFN-gamma on the surface of these cells, since almost all of the cell lines or clones tested (17 of 19) responded to IFN-gamma by the induction or enhancement of the expression for either HLA-DR or HLA-A, -B, and -C (or both). The heterogeneity of induction was also demonstrated between clones derived from a glioma line that did not express HLA-DR after IFN-gamma treatment. The production of HLA-DR by one of the clones was abundant enough to be confirmed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.     

Heterogeneity of radiosensitivity in a human glioma cell line.

Sixteen clones were isolated from an early-passage human glioma cell line (IN859) and have been found to show variation in several biological characteristics including DNA content, modal chromosome number, and morphology. In addition, heterogeneity of radiosensitivity was detected: the doses that gave a surviving fraction of 0.01 varied by a factor of approximately 1.5. The most sensitive (clone 6) and the most resistant (clone 9) clones were selected for further study; their surviving fractions at 2Gy (SF2) were 0.37 and 0.64, respectively. When compared at a fixed radiation dose the sensitive clone surprisingly demonstrated greater split-dose recovery than the resistant clone; it also showed greater low dose-rate sparing.