William K. Dahlberg

In vitro radiosensitivity of human diploid fibroblasts derived from women with unusually sensitive clinical responses to definitive radiation therapy for breast cancer

Between January 1985 and December 1986, 811 patients were treated for carcinoma of the breast at the Joint Center for Radiation Therapy by an identical protocol. Of these 811 patients, five patients (0.6%) were identified as having an unusually sensitive clinical response to routine external beam irradiation. This unusual clinical response was characterized by severe skin erythema and edema during the first few weeks of treatment, requiring treatment breaks. Skin fibroblast cell strains were established from these five women as well as from six women with a normal clinical response to breast irradiation (chosen at random from the population of 811 patients). Radiation survival parameters were determined by a colony formation assay from complete survival curves in coded and blinded samples. Cells from the sensitive patients were significantly more sensitive to the cytotoxic effects of radiation in vitro as determined by the parameters D0, D, D10, and n, than were the strains derived from patients with a normal response. We conclude that an unusually severe response to standard fractionated radiotherapy may be associated with greater intrinsic radiation sensitivity of the individuals somatic cells.

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.

Radiosensitivity in Vitro of Human Soft Tissue Sarcoma Cell Lines and Skin Fibroblasts Derived from the Same Patients

Skin fibroblast cell strains and tumour cell lines were established from 12 patients with various types of soft tissue neoplasms, and radiation survival curve parameters were measured in vitro. Soft tissue sarcoma cells were consistently more sensitive to X-irradiation than fibroblasts isolated from the same patient, and were also more sensitive as a group than cell lines derived from 34 other human tumours. There was a general correlation in radiosensitivity between fibroblasts and tumour cells derived from the same patient, indicating that some component of tumour cell sensitivity may relate to genetic factors in the host. Such genetic factors, however, do not explain all of the heterogeneity in tumour cell response. The response of soft tissue sarcoma in vivo may be dependent on complex radiomodifying factors other than inherent radiation sensitivity, thus making it difficult to predict clinical outcome by use of assays which use survival of irradiated tumour cell lines in vitro as an endpoint.

ATM complexes with HDM2 and promotes its rapid phosphorylation in a p53-independent manner in normal and tumor human cells exposed to ionizing radiation.

To further understand the mechanism(s) by which DNA damage activates p53, we analysed the expression levels of p53 and HDM2 (the human homolog of murine MDM2) in various human diploid fibroblast and tumor cell strains during the period that precedes activation of known downstream effectors of p53. In X-irradiated human cells, HDM2 protein was rapidly phosphorylated in serine/threonine residues in a p53, p14ARF and p73-independent manner. In p53 wild-type cells, HDM2 phosphorylation precedes a detectable increase in the levels of p53 and is not observed in ataxia telangiectasia (AT) fibroblasts. The transfection of AT cells with a vector expressing ATM restored the ability to rapidly phosphorylate HDM2 following X-irradiation, confirming a role for ATM in its phosphorylation. We also show that ATM complexes with HDM2. The DNA lesions signaling the early rapid phosphorylation of HDM2 are a result of X-ray and not UV-type damage. The ATM-promoted early covalent modification of HDM2 in X-irradiated human cells may provide a mechanism to activate p53.

Cellular X-ray repair parameters of early passage squamous cell carcinoma lines derived from patients with known responses to radiotherapy

We have investigated X-ray survival parameters and repair of potentially lethal damage ( PLDR ) in ten early passage squamous cell carcinoma cell lines derived from patients who were biopsied before initiation of radiotherapy or after radiation therapy failure. Radiosensitivity (D0) ranged from 1.07 to 1.93 (Gy), extrapolation numbers (-n) from 1.17 to 2.14 and PLD recovery at 24 h from 1.4 to 20.3. Despite significant differences in these parameters amongst the cell lines, a firm correlation between radiocurability and any individual radiobiological parameter could not be established. Our data suggest that the mechanisms associated with radioresistance are complex and that any single radiobiological parameter may not predict clinical success or failure.

Differing responses of Nijmegen breakage syndrome and ataxia telangiectasia cells to ionizing radiation

Abstract Little, J. B., Nagasawa, H., Dahlberg, W. K., Zdzienicka, M. Z., Burma, S. and Chen, D. J. Differing Responses of Nijmegen Breakage Syndrome and Ataxia Telangiectasia Cells to Ionizing Radiation. Radiat. Res. 158, 319–326 (2002). Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder. Originally thought to be a variant of ataxia telangiectasia (AT), the cellular phenotype of NBS has been described as almost indistinguishable from that of AT. Since the gene involved in NBS has been cloned and its functions studied, we sought to further characterize its cellular phenotype by examining the response of density-inhibited, confluent cultures of human diploid fibroblasts to irradiation in the G0/G1 phase of the cell cycle. Both NBS and AT cells were markedly sensitive to the cytotoxic effects of radiation. NBS cells, however, were proficient in recovery from potentially lethal damage and exhibited a pronounced radiation-induced G1-phase arrest. Irradiated AT cells showed no potentially lethal damage and no G1-phase arrest. Both cell types were hypersensitive to the induction of chromosomal aberrations, whereas the distribution of aberrations in irradiated NBS cells was similar to that of normal controls, AT cells showed a high frequency of chromatid-type aberrations. TP53 and CDKN1A (also known as p21Waf1) expression was attenuated in irradiated NBS cells, but maximal induction occurred 2 h postirradiation, as was observed in normal controls. The similarities and differences in cellular phenotype between irradiated NBS and AT cells are discussed in terms of the functional properties of the signaling pathways downstream of AT involving the NBS1 and TP53 proteins.

Differential response of human and rodent cell lines to chemical inhibition of the repair of potentially lethal damage

SummaryWe have examined the effects of several classes of metabolic inhibitors on the repair of potentially lethal damage in density-inhibited cultures of two rodent and two human cell systems which differ in their growth characteristics. Aphidicolin, 1-β-d-arabinofuranosylcytosine (ara-C) and hydroxyurea showed no effect on PLD repair, whereas the effects of 9-β-d-arabinofuranosyladenine (ara-A) and 3-aminobenzamide (3-AB) were cell line dependent. For example, 3-AB suppressed PLD repair almost completely in CHO cells, but showed no inhibitory effects in human diploid fibroblasts. These results indicate that inhibitors of DNA replication and poly(ADP-ribose) synthesis are not efficient inhibitors of cellular recovery in irradiated cells and, moreover, that such effects may be cell line dependent.

Unexpected sensitivity to radiation of fibroblasts from unaffected parents of children with hereditary retinoblastoma.

The response to ionizing radiation was examined in diploid skin fibroblasts derived from 5 patients with hereditary type retinoblastoma as well as their parents. Unexpected sensitivity to cell killing, as measured by clonogenic survival, as well as enhanced radiation‐induced G1 arrest were observed in at least 1 parental fibroblast strain in all 5 families. In all cases, parental strains were equally or more radiosensitive than the probands. The mutation of the retinoblastoma gene (RB) determined in 4 of 5 probands was either absent from the parental cells, as expected from the negative family histories, or identical, in 1 father who was a known carrier. In the fifth family, the family history was negative for retinoblastoma. We hypothesize that the increased parental cell sensitivity to radiation suggests the presence of an as yet unrecognized genetic event occurring in 1 or both parents of children with retinoblastoma. Whether it increases mutability of the RB locus or other loci or interacts with RB is conjectural.

Differential sensitization of human tumor cells by ara-A to X irradiation and its relationship to inherent radioresponse.

We have previously shown that pretreatment of plateau-phase cultures of human tumor cells with ara-A can markedly sensitize them to the cytotoxic effects of X irradiation; the degree of sensitization varied in two different cell lines. The present study was undertaken to determine whether variability in radiosensitization by ara-A occurs at random in human tumor cell lines or if it is related to their intrinsic radiosensitivity (human tumor radioresponse). The interaction between ara-A and X irradiation was examined in plateau-phase cultures of early-passage tumor cell lines of varying radioresponse (D0 range 0.85-3.15 Gy) subcultured immediately after irradiation to measure survival. In six of the eight cell lines studied, pretreatment with ara-A greatly enhanced the lethal effects of X irradiation in a concentration-dependent fashion. Little or no effect was observed in the two radiosensitive cell lines. When ara-A sensitization was plotted as a function of D10 or D, a linear relationship was observed. These data suggest that pretreatment with ara-A is effective in sensitizing radiation-resistant human tumor cells to the lethal effects of X rays, and that this phenomenon may be dependent upon inherent tumor cell radiosensitivity.