ANIMAL MODEL OF HUMAN DISEASE
Megaloblastic Anemia
Animal Model: Temperature-Sensitive (ts) Mutant Mouse L Cells
Contributed by: Irving Dardick, MD, Department of Laboratory Medicine, Ottawa Civic Hospital, Ottawa K1Y 4E9, Canada; George Setterfield, PhD, Department of Biology, Carleton University, Ottawa KlS 5B6, Canada; and Rose Sheinin, PhD, Department of Microbiology and Parasitology, University of Toronto, Toronto M5S 1Al, Canada.
Biologc Features
Two temperature-sensitive (ts) mutant lines of mouse L cells, ts AlS9 and ts Cl, originally isolated by Thompson et al,1,2 have been utilized to investigate molecular features of DNA svnthesis and the relationship of these events to the gross organization of the interphase nucleus. Both ts mutations block cell and chromosome replication when the cell lines are shifted from the permissive temperature (34 C) to the nonpermissive temperature (38.5 C).5'4 During expression of the mutation in ts AIS9, the cells are capable of synthesizing small single-stranded pieces of DNA but cannot convert the fragments into high-molecular-weight chromosomal DNA.3 Under these conditions, cells accumulate in the G1 to earlv S phase of the cell cycle. The molecular defect in temperatureinhibited ts Cl cells has not been characterized, but the cells are blocked in the late S to C2 phase.5 Cvtologic studies 5,6 reveal similar major changes in cell and nuclear morphology subsequent to expression of both ts mutations. We have shown that when mutant cells are shifted from 34 C (Figure 1) to 38.5 C (Figure 2), there is a substantial increase in cell and nuclear volume and the condensed chromatin masses become fragmented into fine clumps, resulting in the nucleus assuming a markedly homogeneous, dispersed Publication sponsored by the Registry of Comparative Pathology of the Armed Forces Institute of
Pathology and supported bv Public Health Service Grant RR 00301 from the Division of Research
Resources. US Department of Health, Education and Welfare, under the auspices of Universities Associated for Research and Education in Pathology. Inc. Supported by grants from the National Research Council of Canada and the Medical Research Council of Canada. Address reprint requests to Dr. I. Dardick, Department of Laboratory Medicine. Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, Ontario K1Y 4E9, Canada. 849 0002-9440/78/121 1-0849$01.00 ( 1978 American Association of Pathologists
850
DARDICK ET AL
Aerican Journal of Patholog
1-Mutant mouse L cells, ts A1S9, culFgv tured at 34 C for 48 hours Note the compact
alhough fenestrated appearing chromatin within the nucleus (arrow). (Leishman's stain, x 1800)
appearance. These morphologic alterations are reversible following a backshift to 34 C on resumption of semiconservative DNA synthesis.5 Comparison Wh Human Dise Megaloblastic anemia, whether drug-induced or due to folate or vitamin Bu deficiency, occurs as a result of a defect in DNA synthesis; the molecular basis for this defect has not been elucidated. On the basis of recent biochemical data, Hoffbrand et al 7 suggest that the derangement involves accumulation of small fragments of newly synthesized DNA as a result of the inability of the cell to convert these fragments into chromosomal DNA, a situation analogous to the ts A1S9 mutant line. The basis of the characteristic morphologic abnormalities in megaloblastic anemia has been an enigma. However, the changes in nuclear morphology associated with megaloblastic anemia (Figure 3) bear a striking resemblance to the alterations in nuclear structure which occur in the
F.we 2-t Al S9 cells cultured at 38.5 C for 48 hours show a subaal increae in both cell and nuclear size. The chromatin pattern is reticular, and numerous nucleoli with associatd clumps of condensed chromatin are evident (Leishman's stain, x 1800)
Vol. 93, No. 3 December 1978
MEGALOBLASTIC ANEMIA
851
Fgm 3-Bone marrow aspirate from a patent with mnegaobbatc anemia secondary to folate de Note the similarity of the nucler chromatin patten in besophilic (b) and polychromatic (p) mWaobats and ts A1 S9 cells cultured at 38.5 C (Figure 2). (Leishman's stain, .
x
1800)
cultured at 38.5 C (Figure 2). We suggest that the nuclear changes in both cell types result from similar mechanisms initiated by primary defects in DNA synthesis. We have interpreted the cytologic changes in inhibited ts mouse L cells on the basis that in normal cells there may be orderly disaggregationreaggregation cycles occurring within heterochromatin masses associated with chromosome replication. During initiation of DNA synthesis, associated groups of replicons may undergo local disaggregation and then reaggregate following replication. In inhibited ts mouse L cells and in megaloblastic anemia in which DNA synthesis is defective,,'7'6 progressive disaggregation of chromatin and nuclear growth continue as normal but chromatin reaggregation is prevented, resulting in progressive accumulation of finely dispersed chromatin. Defective DNA synthesis resulting in single-stranded regions may also provide explanations for aneuploidy, chromosome gaps and breaks, acentric fragments, and incompletely contracted chromosomes, all of which have been observed in megaloblastic anemia. Cultured cell lines with mutations of the type in ts A1S9 and ts Cl clearly provide excellent material for further experimental study of the relation between biochemical and morphologic events in the cell cycle. ts mutants
852
DARDICK ET AL
American Journal of Pathology
Avaiaity
Samples of the mutant mouse L cells ts AlS9 and ts Cl are available from Dr. R. Sheinin, Department of Microbiology and Parasitology, University of Toronto, 150 College Street, Toronto, Ontario, M5S lAl, Canada. References 1. Thompson LH, Mankovitz R, Baker RM, Till JE, Sirninovitch L, Whitmore GF: Isolation of temperature-sensitive mutants of L-cells. Proc Natl Acad Sci USA 66:377-384, 1970 2. Thompson LH, Mankovitz R, Baker RM, Wright JA, Till JE, Siminovitch L Whitmore GF: Selective and non-selective isolation of temperature-sensitive mutants of mouse L-cells and their characterization. J Cell Physiol 78:431-440, 1971 3. Sheinin R: Preliminarv characterization of the temperature-sensitive defect in DNA replication in a mutant mouse L-cell. Cell 7:49-57, 1976 4. Sheinin R, Guttman S: Semi-conservative and non-conservative replication of DNA in temperature-sensitive mouse L-cells. Biochim Biophys Acta 479:105-118, 1977 5. Setterfield G, Sheinin R, Dardick I, Kiss G, Dubskv M: Structure of interphase nuclei in relation to the cell cycle: Chromosome organization in mouse L-cells temperature-sensitive for DNA replication. J Cell Biol 77:246-263, 1978 6. Dardick I, Sheinin R, Setterfield G: Mutant mouse L-cells: A Model for megaloblastic anemia. Br J Haematol 39:483, 1978 7. Hoffbrand AV, Ganeshaguru K, Hooton JWL, Tripp E: Megaloblastic anemia: Initiation of DNA svnthesis in excess of DNA chain elongation as the underl%ing mechanism. Clin Haematol 5:727-745, 1976 8. Wickramasinghe SN, Cooper EH, Chalmers DG: A studv of ervthropoiesis bv combined morphologic, quantitative cvtochemical and autoradiographic methods: Normal human bone marrow, vitamin Bu deficienc and iron deficiencv anemia. Blood 31:304-313, 1968 9. Kass L: Megaloblastic Anemia. Major Problems in Internal Medicine, Vol 8. Philadelphia, W. B. Saunders Co., 1976, pp 186-237
Megaloblastic Anemia
Animal Model: Temperature-Sensitive (ts) Mutant Mouse L Cells
Contributed by: Irving Dardick, MD, Department of Laboratory Medicine, Ottawa Civic Hospital, Ottawa K1Y 4E9, Canada; George Setterfield, PhD, Department of Biology, Carleton University, Ottawa KlS 5B6, Canada; and Rose Sheinin, PhD, Department of Microbiology and Parasitology, University of Toronto, Toronto M5S 1Al, Canada.
Biologc Features
Two temperature-sensitive (ts) mutant lines of mouse L cells, ts AlS9 and ts Cl, originally isolated by Thompson et al,1,2 have been utilized to investigate molecular features of DNA svnthesis and the relationship of these events to the gross organization of the interphase nucleus. Both ts mutations block cell and chromosome replication when the cell lines are shifted from the permissive temperature (34 C) to the nonpermissive temperature (38.5 C).5'4 During expression of the mutation in ts AIS9, the cells are capable of synthesizing small single-stranded pieces of DNA but cannot convert the fragments into high-molecular-weight chromosomal DNA.3 Under these conditions, cells accumulate in the G1 to earlv S phase of the cell cycle. The molecular defect in temperatureinhibited ts Cl cells has not been characterized, but the cells are blocked in the late S to C2 phase.5 Cvtologic studies 5,6 reveal similar major changes in cell and nuclear morphology subsequent to expression of both ts mutations. We have shown that when mutant cells are shifted from 34 C (Figure 1) to 38.5 C (Figure 2), there is a substantial increase in cell and nuclear volume and the condensed chromatin masses become fragmented into fine clumps, resulting in the nucleus assuming a markedly homogeneous, dispersed Publication sponsored by the Registry of Comparative Pathology of the Armed Forces Institute of
Pathology and supported bv Public Health Service Grant RR 00301 from the Division of Research
Resources. US Department of Health, Education and Welfare, under the auspices of Universities Associated for Research and Education in Pathology. Inc. Supported by grants from the National Research Council of Canada and the Medical Research Council of Canada. Address reprint requests to Dr. I. Dardick, Department of Laboratory Medicine. Ottawa Civic Hospital, 1053 Carling Avenue, Ottawa, Ontario K1Y 4E9, Canada. 849 0002-9440/78/121 1-0849$01.00 ( 1978 American Association of Pathologists
850
DARDICK ET AL
Aerican Journal of Patholog
1-Mutant mouse L cells, ts A1S9, culFgv tured at 34 C for 48 hours Note the compact
alhough fenestrated appearing chromatin within the nucleus (arrow). (Leishman's stain, x 1800)
appearance. These morphologic alterations are reversible following a backshift to 34 C on resumption of semiconservative DNA synthesis.5 Comparison Wh Human Dise Megaloblastic anemia, whether drug-induced or due to folate or vitamin Bu deficiency, occurs as a result of a defect in DNA synthesis; the molecular basis for this defect has not been elucidated. On the basis of recent biochemical data, Hoffbrand et al 7 suggest that the derangement involves accumulation of small fragments of newly synthesized DNA as a result of the inability of the cell to convert these fragments into chromosomal DNA, a situation analogous to the ts A1S9 mutant line. The basis of the characteristic morphologic abnormalities in megaloblastic anemia has been an enigma. However, the changes in nuclear morphology associated with megaloblastic anemia (Figure 3) bear a striking resemblance to the alterations in nuclear structure which occur in the
F.we 2-t Al S9 cells cultured at 38.5 C for 48 hours show a subaal increae in both cell and nuclear size. The chromatin pattern is reticular, and numerous nucleoli with associatd clumps of condensed chromatin are evident (Leishman's stain, x 1800)
Vol. 93, No. 3 December 1978
MEGALOBLASTIC ANEMIA
851
Fgm 3-Bone marrow aspirate from a patent with mnegaobbatc anemia secondary to folate de Note the similarity of the nucler chromatin patten in besophilic (b) and polychromatic (p) mWaobats and ts A1 S9 cells cultured at 38.5 C (Figure 2). (Leishman's stain, .
x
1800)
cultured at 38.5 C (Figure 2). We suggest that the nuclear changes in both cell types result from similar mechanisms initiated by primary defects in DNA synthesis. We have interpreted the cytologic changes in inhibited ts mouse L cells on the basis that in normal cells there may be orderly disaggregationreaggregation cycles occurring within heterochromatin masses associated with chromosome replication. During initiation of DNA synthesis, associated groups of replicons may undergo local disaggregation and then reaggregate following replication. In inhibited ts mouse L cells and in megaloblastic anemia in which DNA synthesis is defective,,'7'6 progressive disaggregation of chromatin and nuclear growth continue as normal but chromatin reaggregation is prevented, resulting in progressive accumulation of finely dispersed chromatin. Defective DNA synthesis resulting in single-stranded regions may also provide explanations for aneuploidy, chromosome gaps and breaks, acentric fragments, and incompletely contracted chromosomes, all of which have been observed in megaloblastic anemia. Cultured cell lines with mutations of the type in ts A1S9 and ts Cl clearly provide excellent material for further experimental study of the relation between biochemical and morphologic events in the cell cycle. ts mutants
852
DARDICK ET AL
American Journal of Pathology
Avaiaity
Samples of the mutant mouse L cells ts AlS9 and ts Cl are available from Dr. R. Sheinin, Department of Microbiology and Parasitology, University of Toronto, 150 College Street, Toronto, Ontario, M5S lAl, Canada. References 1. Thompson LH, Mankovitz R, Baker RM, Till JE, Sirninovitch L, Whitmore GF: Isolation of temperature-sensitive mutants of L-cells. Proc Natl Acad Sci USA 66:377-384, 1970 2. Thompson LH, Mankovitz R, Baker RM, Wright JA, Till JE, Siminovitch L Whitmore GF: Selective and non-selective isolation of temperature-sensitive mutants of mouse L-cells and their characterization. J Cell Physiol 78:431-440, 1971 3. Sheinin R: Preliminarv characterization of the temperature-sensitive defect in DNA replication in a mutant mouse L-cell. Cell 7:49-57, 1976 4. Sheinin R, Guttman S: Semi-conservative and non-conservative replication of DNA in temperature-sensitive mouse L-cells. Biochim Biophys Acta 479:105-118, 1977 5. Setterfield G, Sheinin R, Dardick I, Kiss G, Dubskv M: Structure of interphase nuclei in relation to the cell cycle: Chromosome organization in mouse L-cells temperature-sensitive for DNA replication. J Cell Biol 77:246-263, 1978 6. Dardick I, Sheinin R, Setterfield G: Mutant mouse L-cells: A Model for megaloblastic anemia. Br J Haematol 39:483, 1978 7. Hoffbrand AV, Ganeshaguru K, Hooton JWL, Tripp E: Megaloblastic anemia: Initiation of DNA svnthesis in excess of DNA chain elongation as the underl%ing mechanism. Clin Haematol 5:727-745, 1976 8. Wickramasinghe SN, Cooper EH, Chalmers DG: A studv of ervthropoiesis bv combined morphologic, quantitative cvtochemical and autoradiographic methods: Normal human bone marrow, vitamin Bu deficienc and iron deficiencv anemia. Blood 31:304-313, 1968 9. Kass L: Megaloblastic Anemia. Major Problems in Internal Medicine, Vol 8. Philadelphia, W. B. Saunders Co., 1976, pp 186-237
