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Permanent cell culture lines of rat erythroleukemia and growth inhibition by dexamethasone in vitro
Cancer Letters, 17 (1982) 205-211 Elsevier Scientific Publishers Ireland Ltd.
205
PERMANENT CELL CULTURE LINES OF RAT ERYTHROLEUXEMIA AND GROWTH INHIBITION BY DEXAMETHASONE IN VITRO
NORIFUMI HUGGINS=
UEDAawb , NOZOMU TAKEUCHIa,
RYO FUKUNISHIb
and CHARLES
B.
aCentral Laboratory for Clinical Investigation, Ehime University Hospital, bDepartment of Pathology, Ehime University School of Medicine, Ehime (Japan) and ‘Ben May Laboratory for Cancer Research, The University of Chicago, Chicago, IL 60637 (U.S.A.) (Received 15 June 198 2) (Revised version received 18 August 1982) (Accepted 21 August 1982)
SUMMARY
Four erythroleukemia cell lines were established in vitro from rats with primary leukemias induced by a set of intravenous injections of 7,8,12trimethylbenz[a]anthracene (TMBA). These cell lines were grown in Dulbecco’s medium with 20% inactivated fetal calf serum; they were characterized by specific consistent chromosome abnormalities, related to No. 2 chromosome, which were maintained in culture for more than 1 year. Addition of dexamethasone prevented the cell growth of 2 of the cell lines in this medium but failed to alter the growth rate of the other 2 cell lines. These findings are correlated with karyotype abnormalities involving chromosome No. 2.
INTRODUCTION
In the Long-Evans (L-E) strain of rat, a set of intravenous injections of large doses of polycyclic aromatic hydrocarbons consistently elicited leukemias [l] within 8 weeks in more than 75% of the animals at risk. Whereas several types of leukemia were induced, the predominant type was erythroleukemia [ 21. Permanent cell culture lines derived from a transplanted hemato-sarcoma, originally induced by 7,l Bdimethylbenz [(I] anthracene( DMBA) have been established [3,4]. These cell lines maintained their erythroid nature for prolonged periods after repeated subculture. In these cell lines differentiation was induced by dimethylsulfoxide as is shown by: (a) decreased cell size; (b) appearance of benzidine-positive cells; (c) synthesis of globin. Here we describe the establishment of permanent cell culture lines from 0304-3835/82/0000-0000/$02.75 o 1982 Elsevier Scientific Publishers Ireland Ltd. - Published
and Printed in Ireland.
206
primary rat erythroieukemias induced by TMBA and the influence of dexamethasone on their growth. The injection of large doses of cortisone [5] caused the regression of transplanted lymphosarcoma in mice. A single injection of dexamethasone [ 61 resulted in rapid regression of a transplanted lymphocytic T-cell leukemia involving thymus and lymph nodes of the rat. Dexamethasone induced differentiation [ 71 of mouse myelogenous leukemia in cell culture with the appearance of phagocytes in vitro. Erythroleukemia of rat in many cases undergoes rapid and complete regression [S] following hypophysectomy. MATERIALS
AND METHODS
We studied L-E rats from our inbred closed colony which has been maintained for 18 years. The animals are bred at random inter se rather than by brother X sister mating. A lipid emulsion of TMBA, O.~%W/V, was prepared by the method of Schurr [9]. Female rats were given a set of 4 intravenous injections, in caudal vein, of TMBA, 35 mg/kg, at biweekly intervals beginning at age 50 days; the first injection is denoted day 0. The diagnosis of leukemia was based on histologic evidence obtained by biopsy of liver and spleen performed on day 50-54 and at biweekly intervals thereafter. Establishment of cell lines. The donor rat was killed when the primary erythroleukemia was in its terminal stage as determined by leukemic involvement in liver and spleen from histological section of biopsy. Under ether anesthesia and with asepsis, a sample of liver was removed, minced with scissors and homogenized by hand in a glass Ten-Broeck grinder in Dulbecco’s modified Eagle’s medium (DMEM) without added serum. The cell suspension was centrifuged at 1500 rev./min for 5 min; the sediment was resuspended in the same medium and left standing for 1 h at 4°C. The sediment which formed in this suspension was discarded. The cells in the supernatant fluid were washed 3 times and finally resuspended in DMEM containing 20% fetal calf serum which had been inactivated by heating at 56°C for 1 h. The number of cells was determined by hemocytometer count and the suspension adjusted to contain 5 X lo6 cells/ml. Cultures of these cells were incubated in plastic tissue culture flasks (Falcon Plastics, Oxnard, CA) in 5 ml of medium at 37”C, with 5% CO2 in air and 80-95s relative humidity. The medium was changed without disturbing the cells every 4-6 days until growth was established and passages were begun. The cultures then grew very rapidly. Effect of dexamethasone on cell growth in vitro Dexamethasone (Merck Sharp and Dohme, NJ) was dissolved in DMEM containing 20% inactivated fetal calf serum; the final concentration of dexamethasone was lo-’ M and 10d6 M. For cell culture 5 X lo3 cells per
207
1 ml were suspended in 5 ml of each concentration of dexamethasone in a plastic tissue culture flask.
Cytogenetic studies Cells obtained 24 h following re-feeding (1 X lo6 cells/ml) were treated with colcemid a final concentration, 0.05 pg/ml, for 60 min at 37°C to accumulate cells at metaphase. The chromosome specimens were prepared by a standard method. Chromosomes in cells were analyzed by Seabright’s Giemsa (G-band) method [lo]. RESULTS
Establishment of cell lines Four erythroleukemia cell lines were established by our method; these were designated T-l, T-2, T-3 and T-4. Each cell line was derived from a different donor with leukemia. T-l, T-2 and T-3 have been cultured continuously for more than 1 year; T-4 has been maintained for 9 months. T-l and T-2 cell lines gro.w faster than T-3 and T-4 (medium change was done more.often). All of the cell lines appear to be quite homogenous and cells are spherical in the liquid medium. Most nuclei possess large masses of chromatin; nucleoli are barely visible. The cytoplasma is basophilic and scanty around the nuclei in smears stained by Giemsa.
Cy togenetic studies Chromosome analysis is shown in Table 1. In the rat, the normal number of chromosomes is 42, but in the T-l cell lines the number was mostly 41, in T-2 cell line 43, in T-3 cell line 40 and in T-4 cell line 48 chromosomes per cell. Stability of chromosome number was consistent for 9-12+ months after establishment of the cell lines. Chromosome patterns of each cell line analyzed by Giemsa banding are shown in Figs. l-4. In T-l cell line (Fig. l), No. 2 chromosome was trisomic and 2 of the chromosomes showed elongation as additional subterminal interbanding was observed. Furthermore, one of the No. 12 chromosomes TABLE 1 CHROMOSOME,COMPLEMENT IN ERYTHROLEUKEMIC CELL LINES Leukemia cell line
T-l T-2 T-3 T-4
Cell analyzed
33 35 46 30
Number of chromosomes 39
40
41
42
43
44
1
8
22 2 2
1 2 1
30
1 1
4
39
45
46
1
47 . 48
7
14
49
50
4
4
208
1
II
I# 8
7
8
9
IO
7
13
ar 14
88 15
1;11 16
8ll 13
tt 14
18 19
&(L 20
m 19
lllr 20
2 w
trl
12
3
llrrM(IIicII 7 8 9
13
14
15
4
1
2
10
m 7
HI 8
16
utl 13
14
mu 19
20
a&m
3 19
\k
20
Figs. l-4. Karyotypes of rat erythroleukemias, established as permanent cell culture lines.
4
1
I I I8 x
x
T-l to T-4, respectively, which were
was translocated to No. 9 chromosome and one sex chromosome was missing. In T-2 cell line (Fig. 2), No. 2 chromosome trisomy was also observed and the elongation of one No. 2. The other chromosomes were completely normal. T-3 cell line (Fig. 3) showed diploid stemline, but No. 11 chromosome was translocated to No. 2 and No. 6 chromosome is missing. In T-4 cell line (Fig. 4) No. 2 chromosome is trisomic and many extra chromosomes (unidentified) were observed.
Effect of dexamethasone in vitro Results are shown in Figs. 5 and 6. In T-1 and T-2 cell lines, 2 concentrations of dexamethasone, lo-’ M and low6 M, respectively,‘failed to inhibit cell growth. The rate of cell growth in DMEM containing 20% heat-inactivated fetal calf serum to which dexamethasone was added was the same (Fig. 5) as that of cells in a control flask without dexamethasone. Conversely, T-3 and T-4 cell lines experienced inhibition of cell growth (Fig. 6) when dexamethasone, IO-’ M and lO-‘j M, was added. Growth was not observed in either of these cell lines in DMEM containing 20% inactivated fetal calf serum plus dexamethasone. Microscopically, all of cells in the flasks containing dexamethasone were shrunken. The percentage of benzidinepositive cells was increased. DISCUSSION
It was found to be simple to establish cell culture lines of chemically induced rat erythroleukemias. The establishment of 4 cell lines of leukemia with consistent chromosome abnormalities is described in this paper. These cell lines were established from primary rat leukemias, in advanced stages,
DAY 18
""". 2 3
1
4
5
6
7
”
6
”
”
9 10 11 12 13 14
Fig. 5. In permanent cell line T-l, two concentrations cell growth.
12
3
4
5
6
7
of dexamethasone
6
910111213
failed to inhibit
Fig. 6. In permanent cell line T-3, dexamethasone lo-’ M and 10F6 M completely prevented cell growth. (0) Control medium without dexamethasone; ( l ) medium contained lo-’ M dexamethasone; (a) medium contained dexamethasone 10.’ M. I
210
which had been induced by a set of intravenous injections of TMBA rather than from transplanted erythroid hematosarcoma [ 31. Chromosome abnormalities in primary rat leukemia induced by polycyclic aromatic hydrocarbons have been described [ 11-131. The largest telocentric (No. 2) chromosome is characterized by highly reproducible abnormalities. In cytogenetic studies of our cell lines all show the involvement of the No. 2 chromosome in chromosome changes. We believe this to be a significant cytogenetic observation visa+& cell culture. Since cytogenetic studies of the leukemic rat donors were not carried out before starting to transfer into tissue culture, it is not known whether the chromosome abnormality of these cell lines was a primary change during induction of leukemia or a secondary change during culture. But from previous results [ 141, it is possible that those chromosomal abnormalities were elicited in primary leukemogenesis. Three out of 4 cell lines showed No. 2 chromosome trisomy with or without elongation of chromosome. These results may indicate that cell lines with No. 2 trisomy can easily be established in permanent cell culture. Also, other chromosome changes were observed such as translocations from No. 12 chromosome to No. 9 and from No. 11 to No. 2. In our studies, dexamethasone prevented the growth of 2 rat erythroleukemic cell lines in liquid medium but the growth inhibition effect of dexamethasone on erythroleukemia is not consistent because the growth of 2 other cell lines was not impaired. The difference may be related to chromosome abnormality. The erythroleukemic cell lines with No. 2 chromosome which was normal in length in diploid or trisomy represent leukemia in which there was prevention of growth by dexamethasone. On the contrary, the cell lines with elongation of No. 2 chromosome were not inhibited by dexamethasone. The relationship between the other chromosome abnormalities, except No. 2 chromosome, and the effect of dexamethasone is still not clear; further studies are needed. REFERENCES 1 Huggins, C.B. (1979) Induction and extinction of leukemia in rat. In: Experimental Leukemia and Mammary Cancer, pp. 113-129. University of Chicago Press, Chicago. 2 Huggins, C.B. and Sugiyama, T. (1966) Induction of leukemia in rat by pulse-dose of 7,12-dimethylbenx(a)anthracene. Proc. Natl. Acad. Sci. U.S.A., 55, 74-61. 3 Kluge,N., Ostertag, T., Sugiyama, T., Arndt-Jovin, D., Steinheider, G., Furusawa, M. and Dube, S.K. (1976) Dimethylsulfoxide-induced differentiation and hemoglobin synthesis in tissue cultures of rat erythroleukemia cells transformed by 7 ,12dimethylbenz(a)anthracene. Proc. Natl. Acad. Sci. U.S.A., 73, 1237-1240. 4 Maeda, S., Uenaka, H., Ueda, N., Shiraishi, N. and Sugiyama, T. (1980)Establishment and chromosome studies of in vitro lines of chemically induced rat erythroblastic leukemia cells. J. Natl. Cancer Inst., 64, 539-545. 5 Heilman, F.R. and Kendall, E.C. (1944) The influence of lldehydro-17-hydroxycorticosterone (Compound E) on the growth of a malignant tumor in the mouse. Endocrinology, 34,416-420.
211 6 Huggins, C.B. and Kuwahara, I. (1967) Effect of dexamethasone on stem-cell leukemias of rat. In: Endogenous Factors Influencing Host-Tumor Balance, pp. 9-14. Editors: R.C. WissIer, T.L. Dao and S. Wood, Unversity of Chicago Press, Chicago. 7 Lotem, J. and Sachs, L. (1977) Genetic dessection of the control of normal differentiation in myeloid leukemic cells. Proc. N&l. Acad. Sci. U.S.A., 74, 5554-5558. 8 Huggins, C.B. and Oka, H. (1972) Regression of stem-cell erythroblastic leukemia after hypophysectomy. Cancer Res., 32, 239-242. 9 Schurr, P.E. (1969) Composition and preparation of experimental intravenous fat emulsion. Cancer Res., 29, 258-260. 10 Seabright, M. (1971) A rapid banding technique for human chromosome. Lancet, i, 971-973. 11 Sugiyama, T., Kurita, Y. and Nishizuka, Y. (1967) Chromosome abnormality in rat leukemia induced by 7,12_dimethylbenz(a)anthracene. Science, 158, 1058-1059. 12 Sugiyama, T., Kurita, Y. and Nishizuka, Y. (1969) Biologic studies on 7,12-dimethylbenz(a)anthracene-induced rat leukemia with special reference to the specific chromosomal abnormalities. Cancer Res., 29,1117-1124. 13 Kurita, Y., Sugiyama, T. and Nishizuka, Y. (1969) Cytogenetic analysis of the cell population in rat leukemia induced by pulsedoses of 7 ,12dimethylbenz(a)anthracene. Gann, 60,529-535. 14 Sugiyama, T., Uenaka, H., Ueda, N., Fukuhara, S. and Maeda, S. (1978) Reproducible chromosome changes of polycyclic hydrocarbon-induced rat leukemia: incidence and chromosome banding pattern. J. Natl. Cancer Inst., 60,153-160.
205
PERMANENT CELL CULTURE LINES OF RAT ERYTHROLEUXEMIA AND GROWTH INHIBITION BY DEXAMETHASONE IN VITRO
NORIFUMI HUGGINS=
UEDAawb , NOZOMU TAKEUCHIa,
RYO FUKUNISHIb
and CHARLES
B.
aCentral Laboratory for Clinical Investigation, Ehime University Hospital, bDepartment of Pathology, Ehime University School of Medicine, Ehime (Japan) and ‘Ben May Laboratory for Cancer Research, The University of Chicago, Chicago, IL 60637 (U.S.A.) (Received 15 June 198 2) (Revised version received 18 August 1982) (Accepted 21 August 1982)
SUMMARY
Four erythroleukemia cell lines were established in vitro from rats with primary leukemias induced by a set of intravenous injections of 7,8,12trimethylbenz[a]anthracene (TMBA). These cell lines were grown in Dulbecco’s medium with 20% inactivated fetal calf serum; they were characterized by specific consistent chromosome abnormalities, related to No. 2 chromosome, which were maintained in culture for more than 1 year. Addition of dexamethasone prevented the cell growth of 2 of the cell lines in this medium but failed to alter the growth rate of the other 2 cell lines. These findings are correlated with karyotype abnormalities involving chromosome No. 2.
INTRODUCTION
In the Long-Evans (L-E) strain of rat, a set of intravenous injections of large doses of polycyclic aromatic hydrocarbons consistently elicited leukemias [l] within 8 weeks in more than 75% of the animals at risk. Whereas several types of leukemia were induced, the predominant type was erythroleukemia [ 21. Permanent cell culture lines derived from a transplanted hemato-sarcoma, originally induced by 7,l Bdimethylbenz [(I] anthracene( DMBA) have been established [3,4]. These cell lines maintained their erythroid nature for prolonged periods after repeated subculture. In these cell lines differentiation was induced by dimethylsulfoxide as is shown by: (a) decreased cell size; (b) appearance of benzidine-positive cells; (c) synthesis of globin. Here we describe the establishment of permanent cell culture lines from 0304-3835/82/0000-0000/$02.75 o 1982 Elsevier Scientific Publishers Ireland Ltd. - Published
and Printed in Ireland.
206
primary rat erythroieukemias induced by TMBA and the influence of dexamethasone on their growth. The injection of large doses of cortisone [5] caused the regression of transplanted lymphosarcoma in mice. A single injection of dexamethasone [ 61 resulted in rapid regression of a transplanted lymphocytic T-cell leukemia involving thymus and lymph nodes of the rat. Dexamethasone induced differentiation [ 71 of mouse myelogenous leukemia in cell culture with the appearance of phagocytes in vitro. Erythroleukemia of rat in many cases undergoes rapid and complete regression [S] following hypophysectomy. MATERIALS
AND METHODS
We studied L-E rats from our inbred closed colony which has been maintained for 18 years. The animals are bred at random inter se rather than by brother X sister mating. A lipid emulsion of TMBA, O.~%W/V, was prepared by the method of Schurr [9]. Female rats were given a set of 4 intravenous injections, in caudal vein, of TMBA, 35 mg/kg, at biweekly intervals beginning at age 50 days; the first injection is denoted day 0. The diagnosis of leukemia was based on histologic evidence obtained by biopsy of liver and spleen performed on day 50-54 and at biweekly intervals thereafter. Establishment of cell lines. The donor rat was killed when the primary erythroleukemia was in its terminal stage as determined by leukemic involvement in liver and spleen from histological section of biopsy. Under ether anesthesia and with asepsis, a sample of liver was removed, minced with scissors and homogenized by hand in a glass Ten-Broeck grinder in Dulbecco’s modified Eagle’s medium (DMEM) without added serum. The cell suspension was centrifuged at 1500 rev./min for 5 min; the sediment was resuspended in the same medium and left standing for 1 h at 4°C. The sediment which formed in this suspension was discarded. The cells in the supernatant fluid were washed 3 times and finally resuspended in DMEM containing 20% fetal calf serum which had been inactivated by heating at 56°C for 1 h. The number of cells was determined by hemocytometer count and the suspension adjusted to contain 5 X lo6 cells/ml. Cultures of these cells were incubated in plastic tissue culture flasks (Falcon Plastics, Oxnard, CA) in 5 ml of medium at 37”C, with 5% CO2 in air and 80-95s relative humidity. The medium was changed without disturbing the cells every 4-6 days until growth was established and passages were begun. The cultures then grew very rapidly. Effect of dexamethasone on cell growth in vitro Dexamethasone (Merck Sharp and Dohme, NJ) was dissolved in DMEM containing 20% inactivated fetal calf serum; the final concentration of dexamethasone was lo-’ M and 10d6 M. For cell culture 5 X lo3 cells per
207
1 ml were suspended in 5 ml of each concentration of dexamethasone in a plastic tissue culture flask.
Cytogenetic studies Cells obtained 24 h following re-feeding (1 X lo6 cells/ml) were treated with colcemid a final concentration, 0.05 pg/ml, for 60 min at 37°C to accumulate cells at metaphase. The chromosome specimens were prepared by a standard method. Chromosomes in cells were analyzed by Seabright’s Giemsa (G-band) method [lo]. RESULTS
Establishment of cell lines Four erythroleukemia cell lines were established by our method; these were designated T-l, T-2, T-3 and T-4. Each cell line was derived from a different donor with leukemia. T-l, T-2 and T-3 have been cultured continuously for more than 1 year; T-4 has been maintained for 9 months. T-l and T-2 cell lines gro.w faster than T-3 and T-4 (medium change was done more.often). All of the cell lines appear to be quite homogenous and cells are spherical in the liquid medium. Most nuclei possess large masses of chromatin; nucleoli are barely visible. The cytoplasma is basophilic and scanty around the nuclei in smears stained by Giemsa.
Cy togenetic studies Chromosome analysis is shown in Table 1. In the rat, the normal number of chromosomes is 42, but in the T-l cell lines the number was mostly 41, in T-2 cell line 43, in T-3 cell line 40 and in T-4 cell line 48 chromosomes per cell. Stability of chromosome number was consistent for 9-12+ months after establishment of the cell lines. Chromosome patterns of each cell line analyzed by Giemsa banding are shown in Figs. l-4. In T-l cell line (Fig. l), No. 2 chromosome was trisomic and 2 of the chromosomes showed elongation as additional subterminal interbanding was observed. Furthermore, one of the No. 12 chromosomes TABLE 1 CHROMOSOME,COMPLEMENT IN ERYTHROLEUKEMIC CELL LINES Leukemia cell line
T-l T-2 T-3 T-4
Cell analyzed
33 35 46 30
Number of chromosomes 39
40
41
42
43
44
1
8
22 2 2
1 2 1
30
1 1
4
39
45
46
1
47 . 48
7
14
49
50
4
4
208
1
II
I# 8
7
8
9
IO
7
13
ar 14
88 15
1;11 16
8ll 13
tt 14
18 19
&(L 20
m 19
lllr 20
2 w
trl
12
3
llrrM(IIicII 7 8 9
13
14
15
4
1
2
10
m 7
HI 8
16
utl 13
14
mu 19
20
a&m
3 19
\k
20
Figs. l-4. Karyotypes of rat erythroleukemias, established as permanent cell culture lines.
4
1
I I I8 x
x
T-l to T-4, respectively, which were
was translocated to No. 9 chromosome and one sex chromosome was missing. In T-2 cell line (Fig. 2), No. 2 chromosome trisomy was also observed and the elongation of one No. 2. The other chromosomes were completely normal. T-3 cell line (Fig. 3) showed diploid stemline, but No. 11 chromosome was translocated to No. 2 and No. 6 chromosome is missing. In T-4 cell line (Fig. 4) No. 2 chromosome is trisomic and many extra chromosomes (unidentified) were observed.
Effect of dexamethasone in vitro Results are shown in Figs. 5 and 6. In T-1 and T-2 cell lines, 2 concentrations of dexamethasone, lo-’ M and low6 M, respectively,‘failed to inhibit cell growth. The rate of cell growth in DMEM containing 20% heat-inactivated fetal calf serum to which dexamethasone was added was the same (Fig. 5) as that of cells in a control flask without dexamethasone. Conversely, T-3 and T-4 cell lines experienced inhibition of cell growth (Fig. 6) when dexamethasone, IO-’ M and lO-‘j M, was added. Growth was not observed in either of these cell lines in DMEM containing 20% inactivated fetal calf serum plus dexamethasone. Microscopically, all of cells in the flasks containing dexamethasone were shrunken. The percentage of benzidinepositive cells was increased. DISCUSSION
It was found to be simple to establish cell culture lines of chemically induced rat erythroleukemias. The establishment of 4 cell lines of leukemia with consistent chromosome abnormalities is described in this paper. These cell lines were established from primary rat leukemias, in advanced stages,
DAY 18
""". 2 3
1
4
5
6
7
”
6
”
”
9 10 11 12 13 14
Fig. 5. In permanent cell line T-l, two concentrations cell growth.
12
3
4
5
6
7
of dexamethasone
6
910111213
failed to inhibit
Fig. 6. In permanent cell line T-3, dexamethasone lo-’ M and 10F6 M completely prevented cell growth. (0) Control medium without dexamethasone; ( l ) medium contained lo-’ M dexamethasone; (a) medium contained dexamethasone 10.’ M. I
210
which had been induced by a set of intravenous injections of TMBA rather than from transplanted erythroid hematosarcoma [ 31. Chromosome abnormalities in primary rat leukemia induced by polycyclic aromatic hydrocarbons have been described [ 11-131. The largest telocentric (No. 2) chromosome is characterized by highly reproducible abnormalities. In cytogenetic studies of our cell lines all show the involvement of the No. 2 chromosome in chromosome changes. We believe this to be a significant cytogenetic observation visa+& cell culture. Since cytogenetic studies of the leukemic rat donors were not carried out before starting to transfer into tissue culture, it is not known whether the chromosome abnormality of these cell lines was a primary change during induction of leukemia or a secondary change during culture. But from previous results [ 141, it is possible that those chromosomal abnormalities were elicited in primary leukemogenesis. Three out of 4 cell lines showed No. 2 chromosome trisomy with or without elongation of chromosome. These results may indicate that cell lines with No. 2 trisomy can easily be established in permanent cell culture. Also, other chromosome changes were observed such as translocations from No. 12 chromosome to No. 9 and from No. 11 to No. 2. In our studies, dexamethasone prevented the growth of 2 rat erythroleukemic cell lines in liquid medium but the growth inhibition effect of dexamethasone on erythroleukemia is not consistent because the growth of 2 other cell lines was not impaired. The difference may be related to chromosome abnormality. The erythroleukemic cell lines with No. 2 chromosome which was normal in length in diploid or trisomy represent leukemia in which there was prevention of growth by dexamethasone. On the contrary, the cell lines with elongation of No. 2 chromosome were not inhibited by dexamethasone. The relationship between the other chromosome abnormalities, except No. 2 chromosome, and the effect of dexamethasone is still not clear; further studies are needed. REFERENCES 1 Huggins, C.B. (1979) Induction and extinction of leukemia in rat. In: Experimental Leukemia and Mammary Cancer, pp. 113-129. University of Chicago Press, Chicago. 2 Huggins, C.B. and Sugiyama, T. (1966) Induction of leukemia in rat by pulse-dose of 7,12-dimethylbenx(a)anthracene. Proc. Natl. Acad. Sci. U.S.A., 55, 74-61. 3 Kluge,N., Ostertag, T., Sugiyama, T., Arndt-Jovin, D., Steinheider, G., Furusawa, M. and Dube, S.K. (1976) Dimethylsulfoxide-induced differentiation and hemoglobin synthesis in tissue cultures of rat erythroleukemia cells transformed by 7 ,12dimethylbenz(a)anthracene. Proc. Natl. Acad. Sci. U.S.A., 73, 1237-1240. 4 Maeda, S., Uenaka, H., Ueda, N., Shiraishi, N. and Sugiyama, T. (1980)Establishment and chromosome studies of in vitro lines of chemically induced rat erythroblastic leukemia cells. J. Natl. Cancer Inst., 64, 539-545. 5 Heilman, F.R. and Kendall, E.C. (1944) The influence of lldehydro-17-hydroxycorticosterone (Compound E) on the growth of a malignant tumor in the mouse. Endocrinology, 34,416-420.
211 6 Huggins, C.B. and Kuwahara, I. (1967) Effect of dexamethasone on stem-cell leukemias of rat. In: Endogenous Factors Influencing Host-Tumor Balance, pp. 9-14. Editors: R.C. WissIer, T.L. Dao and S. Wood, Unversity of Chicago Press, Chicago. 7 Lotem, J. and Sachs, L. (1977) Genetic dessection of the control of normal differentiation in myeloid leukemic cells. Proc. N&l. Acad. Sci. U.S.A., 74, 5554-5558. 8 Huggins, C.B. and Oka, H. (1972) Regression of stem-cell erythroblastic leukemia after hypophysectomy. Cancer Res., 32, 239-242. 9 Schurr, P.E. (1969) Composition and preparation of experimental intravenous fat emulsion. Cancer Res., 29, 258-260. 10 Seabright, M. (1971) A rapid banding technique for human chromosome. Lancet, i, 971-973. 11 Sugiyama, T., Kurita, Y. and Nishizuka, Y. (1967) Chromosome abnormality in rat leukemia induced by 7,12_dimethylbenz(a)anthracene. Science, 158, 1058-1059. 12 Sugiyama, T., Kurita, Y. and Nishizuka, Y. (1969) Biologic studies on 7,12-dimethylbenz(a)anthracene-induced rat leukemia with special reference to the specific chromosomal abnormalities. Cancer Res., 29,1117-1124. 13 Kurita, Y., Sugiyama, T. and Nishizuka, Y. (1969) Cytogenetic analysis of the cell population in rat leukemia induced by pulsedoses of 7 ,12dimethylbenz(a)anthracene. Gann, 60,529-535. 14 Sugiyama, T., Uenaka, H., Ueda, N., Fukuhara, S. and Maeda, S. (1978) Reproducible chromosome changes of polycyclic hydrocarbon-induced rat leukemia: incidence and chromosome banding pattern. J. Natl. Cancer Inst., 60,153-160.