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Semi-automated system for monitoring chromosome breakage
320
0th ANNUAL MEETING, MIAMI BEACH, FLA.
Tlfis is part of a larger study that will be subnfitted in partial fulfillment for the degree of Doctor of Philosophy at Fordham University. L.A.E. performed this work during the tenure of a grant from the National Fellowships Fund.
26 MELNYK J. *, G. PERSINGER* AND K. R. CASTLEMAN * *, *City of Hope Medical Center,
Duarte, Calif. ; **Jet Propulsion Laboratories, Pasadena, Calif. (U.S.A,).
Semi-automated system for monitoring chromosome breakage A semi-automated systeln for processing st)ecimens of blood, bone marrow and testes onto slides has been developed which is suitable for studies in chronlosome breakage. The sy:stem is capable of processing up to 576 specimens a day with 5 slides per specimen. A system of analysis is proposed which consist:; of two inicroscope:~ equipped with T.V. cameras coupled to a single T.V. monitor through a single computer. A trained technician will be capable of analyzing metaphase cells displayed rapidly on the monitor at the rate of 3 seconds per cell or IOOO cells per hour. The computer will operate the two stepping stages on the micro~,:copes, a metaphase finder, a display and hold mode for the monitor, a chromosome counting program and a memory for types and number of aberrations per cell. The design and operation of this system will be described.
27 JACOBS, L. j . , AND R. DEMARs, University of Wisconsin, Madison, Wis. 537o6
(U.S.A.). Induction of 8-azaguanine-resistant human diploid fibroblasts by 2-nitrosofluorene 2-Nitrosofluorene is known to be carcinogenic in rats and nmtagenic in bacteria. We have investigated whether 2-nitrosofluorene could increase the frequency of 8azaguanine-resistant variants of diploid, human, foreskin-derived fibroblasts. We used four unrelated fibroblast strains and found that 2-nitlosofluorene concentrations of less than 4.o. IO GM resulted in an average cell survival of lOO%, as determined by cloning efficiency, while the average survival was less than one percent at concentrations greater than 1.5"Io -~ M. The steep concentration dependence of these toxic effects and their persistence for less than fifteen minutes in aqueous medium require that application procedures be rigorously controlled to insure reproducibility. To detect the induction of 8-azaguanine-resistant variants, cells are plated at IOn per 5o m m diameter dish (4.5 cells per m m 'z) and treated in s i t , fifteen hours after plating with medium containing 8o per cent F I o (all F I o used in these studies is hypoxanthine free), 14 per cent fetal calf serum, 6 per cent DMSO, and the appropriate concentration of 2-nitrosofluorene. The nitrosofluorene medium is removed after four hours and replaced with non-selective medium containing F I o plus fetal calf serum. After three days, the cells are put in selective medium containing 85% Fio, I5% calf
0th ANNUAL MEETING, MIAMI BEACH, FLA.
Tlfis is part of a larger study that will be subnfitted in partial fulfillment for the degree of Doctor of Philosophy at Fordham University. L.A.E. performed this work during the tenure of a grant from the National Fellowships Fund.
26 MELNYK J. *, G. PERSINGER* AND K. R. CASTLEMAN * *, *City of Hope Medical Center,
Duarte, Calif. ; **Jet Propulsion Laboratories, Pasadena, Calif. (U.S.A,).
Semi-automated system for monitoring chromosome breakage A semi-automated systeln for processing st)ecimens of blood, bone marrow and testes onto slides has been developed which is suitable for studies in chronlosome breakage. The sy:stem is capable of processing up to 576 specimens a day with 5 slides per specimen. A system of analysis is proposed which consist:; of two inicroscope:~ equipped with T.V. cameras coupled to a single T.V. monitor through a single computer. A trained technician will be capable of analyzing metaphase cells displayed rapidly on the monitor at the rate of 3 seconds per cell or IOOO cells per hour. The computer will operate the two stepping stages on the micro~,:copes, a metaphase finder, a display and hold mode for the monitor, a chromosome counting program and a memory for types and number of aberrations per cell. The design and operation of this system will be described.
27 JACOBS, L. j . , AND R. DEMARs, University of Wisconsin, Madison, Wis. 537o6
(U.S.A.). Induction of 8-azaguanine-resistant human diploid fibroblasts by 2-nitrosofluorene 2-Nitrosofluorene is known to be carcinogenic in rats and nmtagenic in bacteria. We have investigated whether 2-nitrosofluorene could increase the frequency of 8azaguanine-resistant variants of diploid, human, foreskin-derived fibroblasts. We used four unrelated fibroblast strains and found that 2-nitlosofluorene concentrations of less than 4.o. IO GM resulted in an average cell survival of lOO%, as determined by cloning efficiency, while the average survival was less than one percent at concentrations greater than 1.5"Io -~ M. The steep concentration dependence of these toxic effects and their persistence for less than fifteen minutes in aqueous medium require that application procedures be rigorously controlled to insure reproducibility. To detect the induction of 8-azaguanine-resistant variants, cells are plated at IOn per 5o m m diameter dish (4.5 cells per m m 'z) and treated in s i t , fifteen hours after plating with medium containing 8o per cent F I o (all F I o used in these studies is hypoxanthine free), 14 per cent fetal calf serum, 6 per cent DMSO, and the appropriate concentration of 2-nitrosofluorene. The nitrosofluorene medium is removed after four hours and replaced with non-selective medium containing F I o plus fetal calf serum. After three days, the cells are put in selective medium containing 85% Fio, I5% calf