- Email: [email protected]
TRANSMISSION OF IN-VITRO RADIORESISTANCE IN A CANCER-PRONE FAMILY
1335
cyanide levels"have been found in necropsy studies of fire
fatalities.5 The source of cyanide is the hydrogen cyanide gas produced by thermal degradation of complex nitrogen polymers found in modern plastic furnishings and in natural
wool and silk. The massive growth of the will polymer industry makes it likely that hydrogen cyanide z be encountered with increasing frequency in fires. 12 We found that fire survivors without clinical evidence of smoke exposure did not have raised cyanide levels when the subjects’ smoking habits were taken into account. However, several of the survivors who had inhaled a considerable amount of smoke had high blood cyanide concentrations, showing that cyanide inhalation need not necessarily be associated with fatalities only. The dependence of hydrogen cyanide production in fires on a variety of factors, such as materials combusted and temperature of the environment, means that cyanide inhalation in domestic fires could be a very variable occurrence. Several of our patients had near-lethal cyanide levels and most had levels liable to produce serious toxicity. 13 Since the results of our work still in progress agree with those ofAnsell and LewisIIthat the half-life for cyanide elimination is very short (approximately 1 h) the initial exposure levels in our survivors were probably considerably higher than the levels measured on admission. Several of the patients with high cyanide levels were apnoeic and moribund on arrival and required emergency resuscitation followed promptly by assisted ventilation and intensive care, including treatment of metabolic acidosis. In smoke inhalation metabolic acidosis is usually attributed to tissue anoxia due to environmental oxygen deprivation, aggravated by interference of carboxyhaemoglobin with oxygen transport to the tissues.l4 Cyanide poisoning also produces lactic acidosis by causing a change from aerobic to anaerobic metabolism-a change which occurs at much lower blood cyanide levels than those seen in our patients.l55 Cyanide toxicity is therefore likely to be an important contributory cause of tissue hypoxia in severe smoke inhalation injury. We were unable to evaluate the, specific contribution of cyanide poisoning to mortality in the 5 with severe smoke inhalation who died after admission, but their deaths were undoubtedly influenced by a variety of factors including the presence and severity of concomitant burns, of pre-existing cardiorespiratory disease, and of complications associated with assisted ventilation in severely injured patients. Several of our patients had cardiac dysrhythmias, renal failure, neurological sequelae, and subsequent psychiatric sequelae known to be associated with carbon monoxide poisoning alonel6or in combination with cyanide
fabrics such
as
toxicity These observations have important therapeutic implications. There is no rapid screening test for cyanide poisoning since it takes about 2 h to extract cyanide from blood for analysis. None of the cyanide antidotes is free from side-effects, 18so they cannot be given empirically to all housefire survivors. However, carboxyhaemoglobin can be measured quickly and easily, and patients most likely to have high cyanide levels are those with high carboxyhaemoglobin levels. Had cyanide antidotes been used in those of our patients with carboxyhaemoglobin levels above an arbitrary concentration of 15%, the measure would have proved unwarranted in only 1 case (fig. 1), and more importantly no patient with cyanide toxicity would have been missed, even without the use of the nomogram in this particular series. The use of carboxyhaemoglobin level as a marker of inhalation of
other toxic
general
products
terms
show its
use
of combustion has been suggested in but our findings are the first to
previously,19
in
a
specific (cyanide) poisoning.
We thank the Scottish Home and Health Department (ref. K/MRS/50/C267) for its support; Dr R. Watson, University Department of Anaesthesia at Glasgow Royal Infirmary, for his help with the chemical analyses; and Assistant Firemaster W. Kelly of the Strathclyde Fire Brigade for his valuable co-operation. Requests for reprints should be addressed to D. C., Department of. Anaesthesia, Royal Infirmary, Glasgow G4 OSF. REFERENCES 1. Bowes PC. Casualties attributed to toxic gas and smoke at fires: a survey of statistics. Med Sci Law 1976; 16: 104-09. 2. The Upholstered Furniture (Safety) Regulations, S11980/725. London: H.M. Stationery Office, 1980. 3. A guide to the new furniture safety regulations. London: Department of Trade, 1980. 4. Woolley WD. Nitrogen containing products from the thermal decomposition of flexible polyurethane foams. Br Polymer J 1972; 4: 27-43. 5. Symington IS, Anderson RA, Thomson I, Oliver JS, Harland WA. Cyanide exposure in fires. Lancet 1978; ii: 91-92. 6. Zawacki BE, Jung RC, Joyce J, Rincon E. Smoke, burns and the natural history of inhalation injury in fire victims. Ann Surg 1977; 185: 100-10 7. Valentour JC, Aggarwal V, Sunshine I Sensitive gas chromatographic determination of cyanide. Analyt Chem 1974; 46: 924-25. 8. Bartlett D. Pathophysiology of exposure to low concentrations of carbon monoxide. Arch Environ Hlth 1968; 16: 719-27. 9. Douglas CG, Haldane S, Haldane JBS. The laws of combination of haemoglobin with carbon monoxide and oxygen. J Physiol (Lond) 1912; 44: 274-304. 10. Cope O, Rhinelanch FW. The problem of burn shock complicated by pulmonary damage. Ann Surg 1943; 117: 915-28. 11. Ansell M, Lewis FAS. A record of cyanide concentrations found in human organs J Forens Sci 1970; 17: 148-55. 12. Two-part survey of the British Plastics Industry 1970-71. Br Plast 1971; 44 (1): 59-80. 13. Graham DL, Lawson D, TheodoreJ, Robin ED. Acute cyanide poisoning complicated by lactic acidosis and pulmonary oedema. Arch Intern Med 1977; 137: 1051-55. 14. Strohl KP, Feldman NT, Saunders NA, O’Connor N. Carbon monoxide poisoning in fire victims: A reappraisal of prognosis. J Trauma 1980; 20: 78-80. 15. Aitken D, West D, Smith F, et al. Cyanide toxicity following nitroprusside induced hypotension. Can Anaesth Soc J 1977; 24: 651-60 16. Smith JS, Brandon S. Morbidity from acute carbon monoxide poisoning at three-year follow up. Br Med J 1973; i: 318-21 17. Pitt BR, Radford EP, Gurtner GH, Traystman RJ. Interaction of carbon monoxide and cyanide on cerebral circulation and metabolism. Arch Environ Hlth 1979; 34: 354-59. 18. Editorial Which antidote for cyanide? Lancet 1977; ii: 1167. 19. Dyer RF, Esch VH. Polyvinyl chloride toxicity in fires. JAMA 1976; 235: 393-97.
TRANSMISSION OF IN-VITRO RADIORESISTANCE IN A CANCER-PRONE FAMILY N. TORBEN BECH-HANSEN BRENDA M. SELL BEATRICE C. LAMPKIN MALCOLM C.
WILLIAM A. BLATTNER ELISABETH A. MCKEEN JOSEPH F. FRAUMENI, JR PATERSON
Health Sciences
Division, Atomic Energy of Canada Limited, Chalk River, Ontario, Canada; Environmental Epidemiology and Clinical Epidemiology Branches, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, U.S.A.; and Hematology/Oncology Department, Children’s Hospital Medical Center, Cincinnati, Ohio, U.S.A.
Neoplasms of possible radiogenic origin developed in two members of a family prone diversity of cancers, including bone and soft-tissue
Summary to a
sarcoma, brain and breast cancers, and leukaemia. Gammairradiation survival studies in these two patients and three other relatives, but not their spouses, over three generations demonstrated resistance to cell killing. The D10 value (radiation dose required to reduce survival to 10%) was significantly higher for the five radioresistant strains (491±30 rad) than for control cultures (405±18 rad). There was a significant correlation between individual D10 values and D0 survival-curve parameters, indicating that changes in the exponential slope of the survival curves accounted for much
1336
of the increase in D10 values. This novel radiation phenotype manifestation of a basic cellular defect, predisposing to a variety of tumours in family members. Thus in-vitro radioresistance, like radiosensitivity, may be a phenotype of a mechanism that increases cancer risk in could be
a
man.
Introduction THE
autosomal recessive disorders, xeroderma and pigmentosum ataxia-telangiectasia, are associated with an increased risk of cancer, and cells from patients with these disorders have shown in-vitro sensitivity to killing by ultraviolet light and gamma-radiation, respectively. 1,2 Defects in DNA repair pathways have been identified which may be linked to cancer susceptibility in these conditions. Recent studies have also identified in-vitro sensitivity to carcinogens (e.g., radiation) in other genetic disorders and several cancer families.3-12 In studies to define determinants that contribute to cancer risks in man we found that skin fibroblast cultures from one family prone to various neoplasms demonstrated a radioresistance (RR) phenotype over three generations. It is suggested that an alteration in an error-prone DNA repair process could account for this phenotype and the familial predisposition to various cancers,
including radiogenic neoplasms. Family History In the initial
report13 family members with diverse neoplasms
documented, including three each with soft-tissue sarcoma, breast cancer, leukaemia, and brain cancer and one each with osteosarcoma, bilateral malignant neurilemmoma, adenocarcinoma of the lung, and polycythaemia vera. The tumours occurred over six generations of a large kindred in a pattern consistent with an were
incompletely penetrant autosomal dominant gene with pleiotropic effects. A portion of the family tree is shown in the accompanying figure. Since the original report, three additional tumours have been identified in the line of descent : choroid haemangioma of the eye in a distant female relative (at age 62), adenocarcinoma of the lung in her son (at age 55), and osteogenic sarcoma of a lumbar vertebra in the proband’s 15-year-old brother (vI-4). This last tumour arose in the field of radiotherapy administered 12 years earlier for malignant neurilemmoma.
Survival response of fibroblast strains after graded doses of gamma. radiation. The stippled area indicates the range in the mean survival response of five normal strains. The best fit of the lines in the exponential region was established by linear regression analysis of data for control strains 3151-T (lower limit) and 1461-T (upper limit). The number designations in the family pedigree (insert top right) and the scattergram of D,values (insert bottom left) calculated from the survival data are those used previously.1 J Dvalues of the five controls are shown in the scattergram in the order GM38,1461-T, 2270-T, 2650-T, 3151-T, from left to right. Each point represents the mean of two to five experiments, and their standard errors were routinely less than 10%. Points for strains V-7 and V-8 are the means of duplicate tests with the two
independent biopsies. OS, osteogenic sarcoma; ALL, acute lymphoblastic leukaemia; NL, bilateral malignant neurilemmoma; SS, soft-tissue sarcoma; BT, brain tumour; BR, breast cancer; and PV, polycythaemia vera. Symbols m the pedigree: solid, cancer; half solid, polycythaemia vera; open, normal.
Laboratory Methods Cultured fibroblasts from eight family members were studied in a colony-forming assayl4 in parallel with fibroblasts from five clinically normal and unrelated individuals and two patients with ataxia-telangiectasia. All strains were derived from skin-biopsy specimens cultured at Meloy Laboratories, Springfield, Virginia, except for strain GM38 from the Institute for Medical Research, Camden, New Jersey, AT2BE (CRL 1343) from the American Type Culture Collection, and AT4BI from Dr A. M. R. Taylor, University of Birmingham, U.K. -
Monolayer cultures of each strain were harvested and suspended in fresh medium (Ham’s F12 supplemented with 10% fetal-calf serum) at 1 x 105 cells/ml and irradiated with graded doses of6oCo gamma-rays at 4°C, cells were diluted to give an estimated 50 surviving colonies and plated along with gamma-ray-inactivated (5 krad) feeder cells for a total of(6-8) x 104 total cells per plate. The resulting colonies were stained and counted 14-21 days later. Results The family was selected for gamma-irradiation survival studies because of the development of two neoplasms suspected of being radiogenic. (Subject Iv-19, who had
polycythaemia vera, worked in a plant manufacturing heavy between 1952 and 1957, and subject vi-4 was therapeutically exposed.) Values of DIO (radiation dose required to reduce survival to 10%) were 532±17 rad for IV-19 and 496.t Irad for vi-4-both significantly more than normal (405±18 rad) (see table). resistant (p<0-001) Cells from two other family members with cancer-the proband (VI-2) and his father (v-8)-were also significantly more resistant than normal. A paternal aunt (V-10) who is clinically normal at age 30 had the RR phenotype, whereas a paternal aunt with leiomyosarcoma (V-l 1) had a DJO value of 396±23 rad, which is in the normal range. Two family water
in the cancer-prone lineage-the mother of the and the paternal grandfather-had normal DlO proband values. In two cases, separate biopsy specimens from the same individual gave highly concordant DIO values; in one of these cases (v-8) the RR phenotype was seen in biopsy specimens taken before and after diagnosis and therapy for members
not
low-grade astrocytoma.
Radiation
resistance is apparent in terms of both the fraction at different radiation doses and the DlO surviving values calculated from linear regression analysis (see figure).
1337
The difference in DIO between normal and resistant strains is a minimum of 50 rad and an average of84 rad. This difference in mean DJO values for the five RR strains (491±30) and the five normal strains (405±18) is highly significant (p<0’ 001). Furthermore, there was a significant correlation (r= 0-926) between individual DIO values and the corresponding survival-curve parameter Do (inverse of the slope of the exponential region of the survival curve), suggesting that changes in the exponential slope of the survival curves CLINICAL SUMMARY AND DIU VALUES FROM SURVIVAL EXPERIMENTS WITH SKIN FIBROBLASTS FROM EIGHT MEMBERS OF A CANCER-PRONE FAMILY AND CONTROLS
several forms of
and may involve a basic cellular RR. This phenotype could be the process 17 manifesting result of some aberrant error-prone DNA repair process or some cell-cycle perturbation. These possibilities are more attractive than the idea of an inherent dose-modifying factor in view of preliminary experiments indicating that a major component of the initial DNA damage after gammairradiation is the same in normal and RR cells. Investigation of recombinational repair, particularly as it relates to the repair of two-strand lesions (e.g., double-strand breaks or interstrand crosslinks) may help to explain RR.18 Furthermore, it may be relevant that resistance to ultraviolet light in human melanoma cell linesl9 is apparently the expression ofavery efficient post-replication repair system.20 Radioresistance offers an alternative to radiosensitivity as an in-vitro phenotype of mechanisms that increase cancer risk in man. Biochemical and molecular characterisation of the defect underlying the RR phenotype may provide important clues to mechanisms of carcinogenesis. cancer as
We thank Ruth E. Brounstein and Ann Stewart for preparing the and Dr N. E. Gentner and Dr J. J. Mulvihill for their helpful . comments on the manuscript.
manuscript
This work was NOI-CP-9100.
supported
in part
by
NCI Contracts NOI-CP-81002 and
Requests for reprints should be addressed to N. T. B-H., Health Sciences Division, Atomic Energy of Canada Limited, Chalk River, Ontario KOJ IJO, Canada.
REFERENCES
*p
represented the major contribution to the increased DIO values. Do values averaged 135±8 rad, for normal control strains and 129±8 rad for the three cancer-family members with normal survival responses, compared to a mean of 157±13 rad for the five radioresistant strains; the corresponding mean n (ordinate value from extrapolation of the exponential portion of the survival curve to zero dose) values were 2 -· 0±04, 2.5--tO -2, and 2 -3±0’3. Discussion
validity of the RR phenotype in this family was suggested by the consistency in replicate experiments with each strain, by the results from biopsies on the same individuals, and by the presence of the RR phenotype only in the cancer-prone lineage of the family and not in spouses. All family members with RR were either clinically affected or at high risk for cancer (v-10), and two had neoplasms that appeared to be radiation-induced. The diversity oftumours in the family is consistent with the dominantly inherited syndrome, described by Li and Fraumeni, of multiple cancers, including soft-tissue The
and brain tumours, leukaemia.15,16In some members of these families increased susceptibility to environmental carcinogens, including ionising radiation, has been suggested. In the present family, RR was seen in four of five affected members over three generations, but not in spouses, suggesting that RR may be a heritable-marker of cancer susceptibility. Since various tumour types occurred in this family, the heritable mechanism of susceptibility may be common to sarcomas,
breast
carcinoma,
1. Robbins JH, Kraemer KH, Lutzner MA, Festoff BW, Coon HG. Xeroderma pigmentosum: an inherited disease with sun sensitivity, multiple cutaneous neoplasms, and abnormal repair. Ann Intern Med 1974; 80: 221-48. 2. Paterson MC, Smith PJ. Ataxia-telangiectasia: an inherited human disorder involving hypersensitivity to ionising radiation and related DNA-damaging chemicals. Ann Rev Genet 1979; 13: 291-318. 3. Arlett CF, Harcourt SA. Survey of radiosensitivity in a variety of human cell strains. Cancer Res 1980; 40: 926-32. 4. Arlett CF, Lehmann AR. Human disorders showing increased sensitivity to the induction of genetic damage. Ann Rev Genet 1978; 12: 95-115. 5. Bech-Hansen NT, Sell BM, Mulvihill JJ, Paterson MC. The association of in vitro radiosensitivity and cancer in a family with acute myelogenous leukemia. Cancer Res 6.
(in press). Friedberg EC, Ehmann UK, Williams JI. Human DNA repair. Adv Radiat Biol 1979; 8: 85-174.
diseases associated with defective
7. Lewis
PD, Corr JB, Arlett CF, Harcourt SA. Increased sensitivity to gammairradiation of skin fibroblasts in Friedreich’s ataxia. Lancet 1979; ii: 454-75. 8. Little JB, Nove J, Weichselbaum RR. Abnormal sensitivity of diploid skin fibroblasts from a family with Gardner’s syndrome to the lethal effects of X-irradiation, ultraviolet light and mitomycin-C. Mutat Res 1980; 70: 241-50. 9. Paterson MC, Bech-Hansen NT, Smith PJ Heritable radiosensitive and DNA repair deficient disorders in man. In: Seeberg E, ed. Chromosome damage and repair. New York: Plenum Press (in press). 10. Smith PJ, Paterson MC. Rothmund-Thomson syndrome: in-vitro radiosensitivity and defective DNA repair in cultured skin fibroblasts. In: Magee PN, Foti M, Bergbauer PA, eds. Proc 71st Ann Meet Am Ass Cancer Res. Baltimore: Waverly Press, Inc., 1980: 110 (abstract). PJ, Paterson MC, Kraemer KH. In-vitro radiosensitivity in a patient with dermatomyositis and cancer. Lancet 1981; i: 216-17. 12. Weichselbaum RR, Nove J, Little JB. X-ray sensitivity of fifty-three human diploid fibroblast cell strains from patients with characterised genetic disorders. Cancer Res 1980; 40: 920-25. 13. Blattner WA, McGuire DB, Mulvihill JJ, Lampkin BC, Hananian J, Fraumeni JF Jr. Genealogy of cancer in a family. JAMA 1979; 241: 259-61. 14. Paterson MC, Anderson AK, Smith BP, Smith PJ. Enhanced radiosensitivity of 11. Smith
15.
16. 17.
18. 19.
20.
cultured fibroblasts from ataxia-telangiectasia heterozygotes manifested by defective colony-forming ability and reduced DNA repair replication after hypoxic &ggr;-irradiation. Cancer Res 1979; 39: 3725-34. Li FP, Fraumeni JF Jr. Soft-tissue sarcomas, breast cancer and other neoplasms: a familial syndrome? Ann Intern Med 1969; 71: 747-52. Li FP, Fraumeni JF Jr. Rhabdomyosarcoma in children: epidemiologic study and identification of a familial cancer syndrome. J Nat Cancer Inst 1969; 43: 1365-73. Comings DE. A general theory of carcinogenesis. Proc Nat Acad Sci USA 1973; 70: 3324-28. Fletcher HL. Resistance to radiation, recombination repair of DNA and chromosome organisation. Mutat Res 1981; 80: 79-89. Chalmers AH, Lavin M, Atisoontornkul SI, Mansbridge JI, Kidson C. Resistance of human melanoma cells to ultraviolet-radiation. Cancer Res 1976; 36: 1930-34. Lavin M, Chalmers AH, Kidson C. DNA repair and UV resistance in human melanoma. In: Hanawalt PC, Setlow RB, eds. Molecular mechanisms for repair of DNA. New York: Plenum Press, 1975: 817-19.
cyanide levels"have been found in necropsy studies of fire
fatalities.5 The source of cyanide is the hydrogen cyanide gas produced by thermal degradation of complex nitrogen polymers found in modern plastic furnishings and in natural
wool and silk. The massive growth of the will polymer industry makes it likely that hydrogen cyanide z be encountered with increasing frequency in fires. 12 We found that fire survivors without clinical evidence of smoke exposure did not have raised cyanide levels when the subjects’ smoking habits were taken into account. However, several of the survivors who had inhaled a considerable amount of smoke had high blood cyanide concentrations, showing that cyanide inhalation need not necessarily be associated with fatalities only. The dependence of hydrogen cyanide production in fires on a variety of factors, such as materials combusted and temperature of the environment, means that cyanide inhalation in domestic fires could be a very variable occurrence. Several of our patients had near-lethal cyanide levels and most had levels liable to produce serious toxicity. 13 Since the results of our work still in progress agree with those ofAnsell and LewisIIthat the half-life for cyanide elimination is very short (approximately 1 h) the initial exposure levels in our survivors were probably considerably higher than the levels measured on admission. Several of the patients with high cyanide levels were apnoeic and moribund on arrival and required emergency resuscitation followed promptly by assisted ventilation and intensive care, including treatment of metabolic acidosis. In smoke inhalation metabolic acidosis is usually attributed to tissue anoxia due to environmental oxygen deprivation, aggravated by interference of carboxyhaemoglobin with oxygen transport to the tissues.l4 Cyanide poisoning also produces lactic acidosis by causing a change from aerobic to anaerobic metabolism-a change which occurs at much lower blood cyanide levels than those seen in our patients.l55 Cyanide toxicity is therefore likely to be an important contributory cause of tissue hypoxia in severe smoke inhalation injury. We were unable to evaluate the, specific contribution of cyanide poisoning to mortality in the 5 with severe smoke inhalation who died after admission, but their deaths were undoubtedly influenced by a variety of factors including the presence and severity of concomitant burns, of pre-existing cardiorespiratory disease, and of complications associated with assisted ventilation in severely injured patients. Several of our patients had cardiac dysrhythmias, renal failure, neurological sequelae, and subsequent psychiatric sequelae known to be associated with carbon monoxide poisoning alonel6or in combination with cyanide
fabrics such
as
toxicity These observations have important therapeutic implications. There is no rapid screening test for cyanide poisoning since it takes about 2 h to extract cyanide from blood for analysis. None of the cyanide antidotes is free from side-effects, 18so they cannot be given empirically to all housefire survivors. However, carboxyhaemoglobin can be measured quickly and easily, and patients most likely to have high cyanide levels are those with high carboxyhaemoglobin levels. Had cyanide antidotes been used in those of our patients with carboxyhaemoglobin levels above an arbitrary concentration of 15%, the measure would have proved unwarranted in only 1 case (fig. 1), and more importantly no patient with cyanide toxicity would have been missed, even without the use of the nomogram in this particular series. The use of carboxyhaemoglobin level as a marker of inhalation of
other toxic
general
products
terms
show its
use
of combustion has been suggested in but our findings are the first to
previously,19
in
a
specific (cyanide) poisoning.
We thank the Scottish Home and Health Department (ref. K/MRS/50/C267) for its support; Dr R. Watson, University Department of Anaesthesia at Glasgow Royal Infirmary, for his help with the chemical analyses; and Assistant Firemaster W. Kelly of the Strathclyde Fire Brigade for his valuable co-operation. Requests for reprints should be addressed to D. C., Department of. Anaesthesia, Royal Infirmary, Glasgow G4 OSF. REFERENCES 1. Bowes PC. Casualties attributed to toxic gas and smoke at fires: a survey of statistics. Med Sci Law 1976; 16: 104-09. 2. The Upholstered Furniture (Safety) Regulations, S11980/725. London: H.M. Stationery Office, 1980. 3. A guide to the new furniture safety regulations. London: Department of Trade, 1980. 4. Woolley WD. Nitrogen containing products from the thermal decomposition of flexible polyurethane foams. Br Polymer J 1972; 4: 27-43. 5. Symington IS, Anderson RA, Thomson I, Oliver JS, Harland WA. Cyanide exposure in fires. Lancet 1978; ii: 91-92. 6. Zawacki BE, Jung RC, Joyce J, Rincon E. Smoke, burns and the natural history of inhalation injury in fire victims. Ann Surg 1977; 185: 100-10 7. Valentour JC, Aggarwal V, Sunshine I Sensitive gas chromatographic determination of cyanide. Analyt Chem 1974; 46: 924-25. 8. Bartlett D. Pathophysiology of exposure to low concentrations of carbon monoxide. Arch Environ Hlth 1968; 16: 719-27. 9. Douglas CG, Haldane S, Haldane JBS. The laws of combination of haemoglobin with carbon monoxide and oxygen. J Physiol (Lond) 1912; 44: 274-304. 10. Cope O, Rhinelanch FW. The problem of burn shock complicated by pulmonary damage. Ann Surg 1943; 117: 915-28. 11. Ansell M, Lewis FAS. A record of cyanide concentrations found in human organs J Forens Sci 1970; 17: 148-55. 12. Two-part survey of the British Plastics Industry 1970-71. Br Plast 1971; 44 (1): 59-80. 13. Graham DL, Lawson D, TheodoreJ, Robin ED. Acute cyanide poisoning complicated by lactic acidosis and pulmonary oedema. Arch Intern Med 1977; 137: 1051-55. 14. Strohl KP, Feldman NT, Saunders NA, O’Connor N. Carbon monoxide poisoning in fire victims: A reappraisal of prognosis. J Trauma 1980; 20: 78-80. 15. Aitken D, West D, Smith F, et al. Cyanide toxicity following nitroprusside induced hypotension. Can Anaesth Soc J 1977; 24: 651-60 16. Smith JS, Brandon S. Morbidity from acute carbon monoxide poisoning at three-year follow up. Br Med J 1973; i: 318-21 17. Pitt BR, Radford EP, Gurtner GH, Traystman RJ. Interaction of carbon monoxide and cyanide on cerebral circulation and metabolism. Arch Environ Hlth 1979; 34: 354-59. 18. Editorial Which antidote for cyanide? Lancet 1977; ii: 1167. 19. Dyer RF, Esch VH. Polyvinyl chloride toxicity in fires. JAMA 1976; 235: 393-97.
TRANSMISSION OF IN-VITRO RADIORESISTANCE IN A CANCER-PRONE FAMILY N. TORBEN BECH-HANSEN BRENDA M. SELL BEATRICE C. LAMPKIN MALCOLM C.
WILLIAM A. BLATTNER ELISABETH A. MCKEEN JOSEPH F. FRAUMENI, JR PATERSON
Health Sciences
Division, Atomic Energy of Canada Limited, Chalk River, Ontario, Canada; Environmental Epidemiology and Clinical Epidemiology Branches, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, U.S.A.; and Hematology/Oncology Department, Children’s Hospital Medical Center, Cincinnati, Ohio, U.S.A.
Neoplasms of possible radiogenic origin developed in two members of a family prone diversity of cancers, including bone and soft-tissue
Summary to a
sarcoma, brain and breast cancers, and leukaemia. Gammairradiation survival studies in these two patients and three other relatives, but not their spouses, over three generations demonstrated resistance to cell killing. The D10 value (radiation dose required to reduce survival to 10%) was significantly higher for the five radioresistant strains (491±30 rad) than for control cultures (405±18 rad). There was a significant correlation between individual D10 values and D0 survival-curve parameters, indicating that changes in the exponential slope of the survival curves accounted for much
1336
of the increase in D10 values. This novel radiation phenotype manifestation of a basic cellular defect, predisposing to a variety of tumours in family members. Thus in-vitro radioresistance, like radiosensitivity, may be a phenotype of a mechanism that increases cancer risk in could be
a
man.
Introduction THE
autosomal recessive disorders, xeroderma and pigmentosum ataxia-telangiectasia, are associated with an increased risk of cancer, and cells from patients with these disorders have shown in-vitro sensitivity to killing by ultraviolet light and gamma-radiation, respectively. 1,2 Defects in DNA repair pathways have been identified which may be linked to cancer susceptibility in these conditions. Recent studies have also identified in-vitro sensitivity to carcinogens (e.g., radiation) in other genetic disorders and several cancer families.3-12 In studies to define determinants that contribute to cancer risks in man we found that skin fibroblast cultures from one family prone to various neoplasms demonstrated a radioresistance (RR) phenotype over three generations. It is suggested that an alteration in an error-prone DNA repair process could account for this phenotype and the familial predisposition to various cancers,
including radiogenic neoplasms. Family History In the initial
report13 family members with diverse neoplasms
documented, including three each with soft-tissue sarcoma, breast cancer, leukaemia, and brain cancer and one each with osteosarcoma, bilateral malignant neurilemmoma, adenocarcinoma of the lung, and polycythaemia vera. The tumours occurred over six generations of a large kindred in a pattern consistent with an were
incompletely penetrant autosomal dominant gene with pleiotropic effects. A portion of the family tree is shown in the accompanying figure. Since the original report, three additional tumours have been identified in the line of descent : choroid haemangioma of the eye in a distant female relative (at age 62), adenocarcinoma of the lung in her son (at age 55), and osteogenic sarcoma of a lumbar vertebra in the proband’s 15-year-old brother (vI-4). This last tumour arose in the field of radiotherapy administered 12 years earlier for malignant neurilemmoma.
Survival response of fibroblast strains after graded doses of gamma. radiation. The stippled area indicates the range in the mean survival response of five normal strains. The best fit of the lines in the exponential region was established by linear regression analysis of data for control strains 3151-T (lower limit) and 1461-T (upper limit). The number designations in the family pedigree (insert top right) and the scattergram of D,values (insert bottom left) calculated from the survival data are those used previously.1 J Dvalues of the five controls are shown in the scattergram in the order GM38,1461-T, 2270-T, 2650-T, 3151-T, from left to right. Each point represents the mean of two to five experiments, and their standard errors were routinely less than 10%. Points for strains V-7 and V-8 are the means of duplicate tests with the two
independent biopsies. OS, osteogenic sarcoma; ALL, acute lymphoblastic leukaemia; NL, bilateral malignant neurilemmoma; SS, soft-tissue sarcoma; BT, brain tumour; BR, breast cancer; and PV, polycythaemia vera. Symbols m the pedigree: solid, cancer; half solid, polycythaemia vera; open, normal.
Laboratory Methods Cultured fibroblasts from eight family members were studied in a colony-forming assayl4 in parallel with fibroblasts from five clinically normal and unrelated individuals and two patients with ataxia-telangiectasia. All strains were derived from skin-biopsy specimens cultured at Meloy Laboratories, Springfield, Virginia, except for strain GM38 from the Institute for Medical Research, Camden, New Jersey, AT2BE (CRL 1343) from the American Type Culture Collection, and AT4BI from Dr A. M. R. Taylor, University of Birmingham, U.K. -
Monolayer cultures of each strain were harvested and suspended in fresh medium (Ham’s F12 supplemented with 10% fetal-calf serum) at 1 x 105 cells/ml and irradiated with graded doses of6oCo gamma-rays at 4°C, cells were diluted to give an estimated 50 surviving colonies and plated along with gamma-ray-inactivated (5 krad) feeder cells for a total of(6-8) x 104 total cells per plate. The resulting colonies were stained and counted 14-21 days later. Results The family was selected for gamma-irradiation survival studies because of the development of two neoplasms suspected of being radiogenic. (Subject Iv-19, who had
polycythaemia vera, worked in a plant manufacturing heavy between 1952 and 1957, and subject vi-4 was therapeutically exposed.) Values of DIO (radiation dose required to reduce survival to 10%) were 532±17 rad for IV-19 and 496.t Irad for vi-4-both significantly more than normal (405±18 rad) (see table). resistant (p<0-001) Cells from two other family members with cancer-the proband (VI-2) and his father (v-8)-were also significantly more resistant than normal. A paternal aunt (V-10) who is clinically normal at age 30 had the RR phenotype, whereas a paternal aunt with leiomyosarcoma (V-l 1) had a DJO value of 396±23 rad, which is in the normal range. Two family water
in the cancer-prone lineage-the mother of the and the paternal grandfather-had normal DlO proband values. In two cases, separate biopsy specimens from the same individual gave highly concordant DIO values; in one of these cases (v-8) the RR phenotype was seen in biopsy specimens taken before and after diagnosis and therapy for members
not
low-grade astrocytoma.
Radiation
resistance is apparent in terms of both the fraction at different radiation doses and the DlO surviving values calculated from linear regression analysis (see figure).
1337
The difference in DIO between normal and resistant strains is a minimum of 50 rad and an average of84 rad. This difference in mean DJO values for the five RR strains (491±30) and the five normal strains (405±18) is highly significant (p<0’ 001). Furthermore, there was a significant correlation (r= 0-926) between individual DIO values and the corresponding survival-curve parameter Do (inverse of the slope of the exponential region of the survival curve), suggesting that changes in the exponential slope of the survival curves CLINICAL SUMMARY AND DIU VALUES FROM SURVIVAL EXPERIMENTS WITH SKIN FIBROBLASTS FROM EIGHT MEMBERS OF A CANCER-PRONE FAMILY AND CONTROLS
several forms of
and may involve a basic cellular RR. This phenotype could be the process 17 manifesting result of some aberrant error-prone DNA repair process or some cell-cycle perturbation. These possibilities are more attractive than the idea of an inherent dose-modifying factor in view of preliminary experiments indicating that a major component of the initial DNA damage after gammairradiation is the same in normal and RR cells. Investigation of recombinational repair, particularly as it relates to the repair of two-strand lesions (e.g., double-strand breaks or interstrand crosslinks) may help to explain RR.18 Furthermore, it may be relevant that resistance to ultraviolet light in human melanoma cell linesl9 is apparently the expression ofavery efficient post-replication repair system.20 Radioresistance offers an alternative to radiosensitivity as an in-vitro phenotype of mechanisms that increase cancer risk in man. Biochemical and molecular characterisation of the defect underlying the RR phenotype may provide important clues to mechanisms of carcinogenesis. cancer as
We thank Ruth E. Brounstein and Ann Stewart for preparing the and Dr N. E. Gentner and Dr J. J. Mulvihill for their helpful . comments on the manuscript.
manuscript
This work was NOI-CP-9100.
supported
in part
by
NCI Contracts NOI-CP-81002 and
Requests for reprints should be addressed to N. T. B-H., Health Sciences Division, Atomic Energy of Canada Limited, Chalk River, Ontario KOJ IJO, Canada.
REFERENCES
*p
represented the major contribution to the increased DIO values. Do values averaged 135±8 rad, for normal control strains and 129±8 rad for the three cancer-family members with normal survival responses, compared to a mean of 157±13 rad for the five radioresistant strains; the corresponding mean n (ordinate value from extrapolation of the exponential portion of the survival curve to zero dose) values were 2 -· 0±04, 2.5--tO -2, and 2 -3±0’3. Discussion
validity of the RR phenotype in this family was suggested by the consistency in replicate experiments with each strain, by the results from biopsies on the same individuals, and by the presence of the RR phenotype only in the cancer-prone lineage of the family and not in spouses. All family members with RR were either clinically affected or at high risk for cancer (v-10), and two had neoplasms that appeared to be radiation-induced. The diversity oftumours in the family is consistent with the dominantly inherited syndrome, described by Li and Fraumeni, of multiple cancers, including soft-tissue The
and brain tumours, leukaemia.15,16In some members of these families increased susceptibility to environmental carcinogens, including ionising radiation, has been suggested. In the present family, RR was seen in four of five affected members over three generations, but not in spouses, suggesting that RR may be a heritable-marker of cancer susceptibility. Since various tumour types occurred in this family, the heritable mechanism of susceptibility may be common to sarcomas,
breast
carcinoma,
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