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Growth rate determines activity of porphobilinogen deaminase both in nonmalignant and malignant cell lines
BIOCHEMICAL
MEDICINE
AND
METABOLIC
40, 213-217
BIOLOGY
(1988)
Growth Rate Determines Activity of Porphobilinogen Deaminase Both in Nonmalignant and Malignant Cell Lines NILI
SCHOENFELD,*
RIVKA YAEL
Laboratory
MAMET,
TEITZ,$
* LEONARD AND
ABRAHAM
LEIBOVICI,~
ORIT
EPSTEIN,*
ATSMON*
of Biochemical Pharmncology, fDepartment of Internul Medicine B. Beilinson Center, 49 100 Petah Tikva, Israel, and #Laborutory of Virology, Sackler Faculty Medicine, Tel Aviv Uni\*ersity. Rumat Aviv. Israel Received
Medical of
June 2. 1988
Porphobilinogen deaminase (PBGD, EC 4.3.1.8), one of the enzymes of the heme biosynthetic pathway, is increased in erythrocytes of patients in the preleukemic state (1) and in patients with malignant lymphoprolipherative disorders (2). In the latter, the enzyme in lymphocytes is also increased (3-5). Malignant transformation of various cell lines by retrograde, transforming viruses and other means also leads to increased activity of PBGD (6). On the other hand, the activity of PBGD was shown to be increased in the fast replicating cells of the regenerating rat liver (7). It was, therefore, unclear whether the increase in PBGD activity was caused by the transforming malignant process or by the increased growth rate induced by this process. In order to further investigate this problem, experiments were carried out in different systems with different growth rates and their PBGD activity was measured. The aim of this study was to determine whether changes in PBGD activity are properties inherent and specific to the malignant transforming event or are related to alterations in growth rate. MATERIALS
AND METHODS
Monolayers of rat embryo fibroblasts, NIH-3T3 cells (rat embryo immortalized fibroblasts which display contact inhibition), L-929 mouse lymphosarcoma cell line, a nontransformed fibroblastic cell line and a MLV (Moloney leukemia virus)and MS (8)-fibroblastic cell line were grown in 25cm’ flasks, in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. Infection of cell lines was proven by high activity of reverse transcriptase measured according to Teitz et al. (8). Transformation was assayed by the “cloning efficiency” method in semisolid agar as described by Bakhanoshvili et al. (9). Cells were considered transformed if 2 x 10’ cells in the inoculum sufficed for colony appearance. 213 0885-4505/88
$3.00
CopyrIght D IYXX by Academic Press. Inc. All right\ of reproduction in any form reserved.
214
SCHOENFELD
ET AL.
Human peripheral mononuclear cells (PMNC) were separated according to Boyum (10). PMNC suspensions, lo6 cells/ml, were kept in petri dishes, 5 cm in diameter, in CITTL medium. CITTL contains DMEM:Modified Ham’s F-12, 1: 1, enriched with casein, insulin, transferrin, testosterone, and linoleic acid (11). Protein was determined according to Lowry et al. (12) with bovine serum albumin as a standard, and DNA according to Boer (13). The incorporation of [14C]thymidine into DNA was measured as previously described (14). The method of Magnussen et al. (15) with slight modifications (4) was employed for the determination of PBGD activity. Student’s t test and Pearson’s product-moment correlation test were used for statistical evaluation. The media DMEM and Ham’s F-12 were purchased from Biological Industries, Kibbutz Beth Haemek, Israel. Human p-cell growth factor (GF) was obtained from The Israel Institute for Biological Research, Ness-Ziona, Israel. Phytohemagglutinin (PA) was a product of Difco Laboratories, Detroit, Michigan, U.S.A. Histopaque was purchased from Sigma Chemical Co., St. Louis, Missouri, U.S.A. All other reagents were of the highest purity available. RESULTS AND DISCUSSION PBGD activity and total protein/flask were determined daily for 7 days in monolayers of rat embryo fibroblasts, NIH-3T3 fibroblasts, and in a mouse lymphosarcoma cell line, L-929. The results are shown in Fig. 1. The figure shows that PBGD activity is positively correlated to the rate of replication of the cells, (total protein/flask) in each of the cell lines examined (P < 0.001). Moreover, increase in PBGD activity is directly related to this rate, the fastest expanding monolayers having the highest PBGD activity. Several points are of special interest: The NIH-3T3 monolayer replicated rapidly until
2%
2.4-
DAYS
FIG. 1. Porphobilinogen deaminase activity and growth rate in various cell lines. Each point in the figure is the mean of three separate determinations of cultures of different cell lines. I. Fibroblasts: protein (0), PBGD (0); II. NIH-3T3: protein (A) PBGD (A); III. L-929: protein (Cl), PBGD (W).
GROWTH
RATE DETERMINES
215
PBGD ACTIVITY
confluence was reached on the fifth day; PBGD activity reached its zenith on the same day and then declined sharply. Though initially the 3T3 cells and the embryo rat fibroblasts had similar PBGD activity, the rate of replication of the former was much faster and their final density in the monolayer was much more marked than that of the embryonal fibroblasts. In accordance with these different growth rates the highest measured PBGD activity of the 3T3 cells was about twice that of the embryonal fibroblasts. The malignant L-929 cells had a higher initial PBGD activity than the two fibroblastic cell lines. Their rate of replication was considerably faster than that of the 3T3 cells and their PBGD activity increased much more than that of the 3T3 cells. When their growth rate declined PBGD activity dropped precipitously. It should be pointed out that, as expected, total DNA/flask, measured daily, correlated positively and significantly with the total amount of protein/plate, P c 0.001. In order to examine the influence on PBGD activity caused by changing the rate of replication of normal cells, PMNC from normal volunteers ‘were treated with P-cell GF and with PA. The results are shown in Table 1. The data in Table 1 show that incubation of the PMNC with GF or PA increased the synthesis of proteins and the incorporation of [14C]-thymidine into DNA, as well as PBGD activity. PA increased [14C]-thymidine incorporation into DNA 65fold and PBGD activity 4-fold. GF, which tripled DNA synthesis, doubled PBGD activity, indicating a positive correlation between rate of growth and PBGD activity. Another set of experiments was carried out in two cell lines with similar growth rates: a nontransformed rat fibroblastic cell line after the 21st passage and an MLV/MS malignantly transformed rat fibroblastic cell line after the 42nd passage (Fig. 2). Malignancy was proven by the cloning efficiency method. As shown in Fig. 2, although the initial PBGD activity was higher in the transformed cell line, the pattern of its activity during 4 days of culture did not differ from the pattern of activity in the nontransformed cells. The above data indicate that, in the systems examined and under the in vitro TABLE 1 The Influence of Growth Factors on PBGD Activity of Human Peripheral Mononuclear
Treatment None GF PA
Protein (mg/dish) 0.19 * 0.02 0.24 r 0.03 N.S. 0.37 * 0.04 P < 0.001
[‘4C]Thymidine incorporation into DNA (cpm/dish) 58 2 17 185 2 18 P < 0.001 387 2 33 P < 0.001
Cells”
PBGD activity (pmol porph/mg prot/hr) 4.5 ” 10.5 * P < 19.2 r P <
1.2 2.1 0.001 2.4 0.001
” Lymphocytes were kept in suspensions as described under Materials and Methods, in the presence of either 50 pg/ml phytohemagglutinin or 10% (v/v) p-cell growth factor. After incubation for 2 days, the various determinations were carried out. The data shown are the means and standard deviations of four separate determinations. N.S., nonsignificant, P > 0.05.
216
SCHOENFELD
ET AL.
FIG. 2. Growth rates and PBGD activities in nontransformed and MLV/MS transformed fibroblastic cell lines. Total protein in each dish was measured daily during the 4 consecutive days of the assay. Total protein on Day I was 0.15 2 0.05 mg protein/dish in both nontransformed and transformed cells. Rate of growth was calculated as mg protein/dish on the indicated day divided by mg protein/dish on the preceding day. In nontransformed fibroblasts (O), in transformed fibroblasts (0). PBGD activity in nontransformed (A) and in transformed fibroblasts (A). The values shown are the results and standard deviations of four separate determinations.
conditions described, changes in PBGD activity are apparently in rate of cell replication and not to malignant processes.
related to changes
SUMMARY
PBGD activity and growth rate were determined in cultures of rat embryo fibroblasts, nontransformed and MLV/MS transformed fibroblastic cell lines; NIH-3T3 cells, and in a mouse lymphosarcoma cell line [L-929]. The two parameters examined correlate positively (P < 0.001). The results of this investigation would seem to indicate clearly that porphobilinogen deaminase activity is related to growth. However, these experiments do not rule out the possibility that malignant transformation per se also causes changes in porphobilinogen deaminase activity. ACKNOWLEDGMENTS We thank Mrs. R. Mevasser for her excellent technical assistance and Mr. and Mrs. D. Sala for their financial support.
REFERENCES I. Pasanen, A. V. O., Vuopio, P., Borgstrom, G. H., and Tenhunen, R., &and. J. Haematol. 27, 35 (1981). 2. Epstein, O., Lahav, M., Schoenfeld, N., Nemesh, L., Shaklai, M., and Atsmon, A., Cancer 52, 828 (1983). 3. Lahav, M., Epstein, O., Schoenfeld, N., Shaklai, M., and Atsmon, A., in “Malignant Lymphomas and Hodgkin’s Disease: Experimental and Therapeutic Advances” (F. Cavali, G. Bonadonna, and M. Rozencweig, Eds.), p. 171. Nijhoff, Boston, 1985. 4. Lahav, M., Epstein, O., Schoenfeld, N., Shaklai, M., and Atsmon, A., J. Amer. Med. Assoc. 257, 39 (1986). 5. Inbal, A., Modan, M.. Weitz, Z., Lahav. M., Schoenfeld, N., Atsmon, A., and Shaklai, M., Cancer 59, 89 (I 987). 6. Teitz. Y., Epstein, 0.. Schoenfeld. N., and Atsmon, A., Med. Sci. Res. 16, 627 (1988).
GROWTH
RATE DETERMINES
PBGD ACTIVITY
217
7. Schoenfeld, N., Mamet, R., Epstein, 0.. Lahav, M., Lurie, Y., and Atsmon, A., Eur. J. Biochem. 166, 663 (1987). 8. Teitz, Y., Lennette, E. H., Oshiro, I. S., and Cremer, N., .I. N&l. Cancer Inst. 46, II (1971). 9. Bakhanoshvili, M., Wrescher, D. H., and Salzberg, S., Cancer Res. 43, 1289 (1983). 10. Boyum, A., Stand. .I. C/in. Lab. Invest. 97 (Suppl 21), 77 (1968). 11. Dartler, F. J., and Insel, P. A., Exp. Cell Res. 138, 287 (1982). 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). 13. Boer, G. J., Anal. Biochem. 65, 225 (1975). 14. Sassa, S., Zalar, G. L., and Kappas, A., J. C/in. Invest. 61, 499 (1978). 15. Magnussen, C. R., Levine, J. B., Doherty, J. M., Cheesman, J. O., and Tschudy, D. P., BIood 44, 857 (1974).
MEDICINE
AND
METABOLIC
40, 213-217
BIOLOGY
(1988)
Growth Rate Determines Activity of Porphobilinogen Deaminase Both in Nonmalignant and Malignant Cell Lines NILI
SCHOENFELD,*
RIVKA YAEL
Laboratory
MAMET,
TEITZ,$
* LEONARD AND
ABRAHAM
LEIBOVICI,~
ORIT
EPSTEIN,*
ATSMON*
of Biochemical Pharmncology, fDepartment of Internul Medicine B. Beilinson Center, 49 100 Petah Tikva, Israel, and #Laborutory of Virology, Sackler Faculty Medicine, Tel Aviv Uni\*ersity. Rumat Aviv. Israel Received
Medical of
June 2. 1988
Porphobilinogen deaminase (PBGD, EC 4.3.1.8), one of the enzymes of the heme biosynthetic pathway, is increased in erythrocytes of patients in the preleukemic state (1) and in patients with malignant lymphoprolipherative disorders (2). In the latter, the enzyme in lymphocytes is also increased (3-5). Malignant transformation of various cell lines by retrograde, transforming viruses and other means also leads to increased activity of PBGD (6). On the other hand, the activity of PBGD was shown to be increased in the fast replicating cells of the regenerating rat liver (7). It was, therefore, unclear whether the increase in PBGD activity was caused by the transforming malignant process or by the increased growth rate induced by this process. In order to further investigate this problem, experiments were carried out in different systems with different growth rates and their PBGD activity was measured. The aim of this study was to determine whether changes in PBGD activity are properties inherent and specific to the malignant transforming event or are related to alterations in growth rate. MATERIALS
AND METHODS
Monolayers of rat embryo fibroblasts, NIH-3T3 cells (rat embryo immortalized fibroblasts which display contact inhibition), L-929 mouse lymphosarcoma cell line, a nontransformed fibroblastic cell line and a MLV (Moloney leukemia virus)and MS (8)-fibroblastic cell line were grown in 25cm’ flasks, in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. Infection of cell lines was proven by high activity of reverse transcriptase measured according to Teitz et al. (8). Transformation was assayed by the “cloning efficiency” method in semisolid agar as described by Bakhanoshvili et al. (9). Cells were considered transformed if 2 x 10’ cells in the inoculum sufficed for colony appearance. 213 0885-4505/88
$3.00
CopyrIght D IYXX by Academic Press. Inc. All right\ of reproduction in any form reserved.
214
SCHOENFELD
ET AL.
Human peripheral mononuclear cells (PMNC) were separated according to Boyum (10). PMNC suspensions, lo6 cells/ml, were kept in petri dishes, 5 cm in diameter, in CITTL medium. CITTL contains DMEM:Modified Ham’s F-12, 1: 1, enriched with casein, insulin, transferrin, testosterone, and linoleic acid (11). Protein was determined according to Lowry et al. (12) with bovine serum albumin as a standard, and DNA according to Boer (13). The incorporation of [14C]thymidine into DNA was measured as previously described (14). The method of Magnussen et al. (15) with slight modifications (4) was employed for the determination of PBGD activity. Student’s t test and Pearson’s product-moment correlation test were used for statistical evaluation. The media DMEM and Ham’s F-12 were purchased from Biological Industries, Kibbutz Beth Haemek, Israel. Human p-cell growth factor (GF) was obtained from The Israel Institute for Biological Research, Ness-Ziona, Israel. Phytohemagglutinin (PA) was a product of Difco Laboratories, Detroit, Michigan, U.S.A. Histopaque was purchased from Sigma Chemical Co., St. Louis, Missouri, U.S.A. All other reagents were of the highest purity available. RESULTS AND DISCUSSION PBGD activity and total protein/flask were determined daily for 7 days in monolayers of rat embryo fibroblasts, NIH-3T3 fibroblasts, and in a mouse lymphosarcoma cell line, L-929. The results are shown in Fig. 1. The figure shows that PBGD activity is positively correlated to the rate of replication of the cells, (total protein/flask) in each of the cell lines examined (P < 0.001). Moreover, increase in PBGD activity is directly related to this rate, the fastest expanding monolayers having the highest PBGD activity. Several points are of special interest: The NIH-3T3 monolayer replicated rapidly until
2%
2.4-
DAYS
FIG. 1. Porphobilinogen deaminase activity and growth rate in various cell lines. Each point in the figure is the mean of three separate determinations of cultures of different cell lines. I. Fibroblasts: protein (0), PBGD (0); II. NIH-3T3: protein (A) PBGD (A); III. L-929: protein (Cl), PBGD (W).
GROWTH
RATE DETERMINES
215
PBGD ACTIVITY
confluence was reached on the fifth day; PBGD activity reached its zenith on the same day and then declined sharply. Though initially the 3T3 cells and the embryo rat fibroblasts had similar PBGD activity, the rate of replication of the former was much faster and their final density in the monolayer was much more marked than that of the embryonal fibroblasts. In accordance with these different growth rates the highest measured PBGD activity of the 3T3 cells was about twice that of the embryonal fibroblasts. The malignant L-929 cells had a higher initial PBGD activity than the two fibroblastic cell lines. Their rate of replication was considerably faster than that of the 3T3 cells and their PBGD activity increased much more than that of the 3T3 cells. When their growth rate declined PBGD activity dropped precipitously. It should be pointed out that, as expected, total DNA/flask, measured daily, correlated positively and significantly with the total amount of protein/plate, P c 0.001. In order to examine the influence on PBGD activity caused by changing the rate of replication of normal cells, PMNC from normal volunteers ‘were treated with P-cell GF and with PA. The results are shown in Table 1. The data in Table 1 show that incubation of the PMNC with GF or PA increased the synthesis of proteins and the incorporation of [14C]-thymidine into DNA, as well as PBGD activity. PA increased [14C]-thymidine incorporation into DNA 65fold and PBGD activity 4-fold. GF, which tripled DNA synthesis, doubled PBGD activity, indicating a positive correlation between rate of growth and PBGD activity. Another set of experiments was carried out in two cell lines with similar growth rates: a nontransformed rat fibroblastic cell line after the 21st passage and an MLV/MS malignantly transformed rat fibroblastic cell line after the 42nd passage (Fig. 2). Malignancy was proven by the cloning efficiency method. As shown in Fig. 2, although the initial PBGD activity was higher in the transformed cell line, the pattern of its activity during 4 days of culture did not differ from the pattern of activity in the nontransformed cells. The above data indicate that, in the systems examined and under the in vitro TABLE 1 The Influence of Growth Factors on PBGD Activity of Human Peripheral Mononuclear
Treatment None GF PA
Protein (mg/dish) 0.19 * 0.02 0.24 r 0.03 N.S. 0.37 * 0.04 P < 0.001
[‘4C]Thymidine incorporation into DNA (cpm/dish) 58 2 17 185 2 18 P < 0.001 387 2 33 P < 0.001
Cells”
PBGD activity (pmol porph/mg prot/hr) 4.5 ” 10.5 * P < 19.2 r P <
1.2 2.1 0.001 2.4 0.001
” Lymphocytes were kept in suspensions as described under Materials and Methods, in the presence of either 50 pg/ml phytohemagglutinin or 10% (v/v) p-cell growth factor. After incubation for 2 days, the various determinations were carried out. The data shown are the means and standard deviations of four separate determinations. N.S., nonsignificant, P > 0.05.
216
SCHOENFELD
ET AL.
FIG. 2. Growth rates and PBGD activities in nontransformed and MLV/MS transformed fibroblastic cell lines. Total protein in each dish was measured daily during the 4 consecutive days of the assay. Total protein on Day I was 0.15 2 0.05 mg protein/dish in both nontransformed and transformed cells. Rate of growth was calculated as mg protein/dish on the indicated day divided by mg protein/dish on the preceding day. In nontransformed fibroblasts (O), in transformed fibroblasts (0). PBGD activity in nontransformed (A) and in transformed fibroblasts (A). The values shown are the results and standard deviations of four separate determinations.
conditions described, changes in PBGD activity are apparently in rate of cell replication and not to malignant processes.
related to changes
SUMMARY
PBGD activity and growth rate were determined in cultures of rat embryo fibroblasts, nontransformed and MLV/MS transformed fibroblastic cell lines; NIH-3T3 cells, and in a mouse lymphosarcoma cell line [L-929]. The two parameters examined correlate positively (P < 0.001). The results of this investigation would seem to indicate clearly that porphobilinogen deaminase activity is related to growth. However, these experiments do not rule out the possibility that malignant transformation per se also causes changes in porphobilinogen deaminase activity. ACKNOWLEDGMENTS We thank Mrs. R. Mevasser for her excellent technical assistance and Mr. and Mrs. D. Sala for their financial support.
REFERENCES I. Pasanen, A. V. O., Vuopio, P., Borgstrom, G. H., and Tenhunen, R., &and. J. Haematol. 27, 35 (1981). 2. Epstein, O., Lahav, M., Schoenfeld, N., Nemesh, L., Shaklai, M., and Atsmon, A., Cancer 52, 828 (1983). 3. Lahav, M., Epstein, O., Schoenfeld, N., Shaklai, M., and Atsmon, A., in “Malignant Lymphomas and Hodgkin’s Disease: Experimental and Therapeutic Advances” (F. Cavali, G. Bonadonna, and M. Rozencweig, Eds.), p. 171. Nijhoff, Boston, 1985. 4. Lahav, M., Epstein, O., Schoenfeld, N., Shaklai, M., and Atsmon, A., J. Amer. Med. Assoc. 257, 39 (1986). 5. Inbal, A., Modan, M.. Weitz, Z., Lahav. M., Schoenfeld, N., Atsmon, A., and Shaklai, M., Cancer 59, 89 (I 987). 6. Teitz. Y., Epstein, 0.. Schoenfeld. N., and Atsmon, A., Med. Sci. Res. 16, 627 (1988).
GROWTH
RATE DETERMINES
PBGD ACTIVITY
217
7. Schoenfeld, N., Mamet, R., Epstein, 0.. Lahav, M., Lurie, Y., and Atsmon, A., Eur. J. Biochem. 166, 663 (1987). 8. Teitz, Y., Lennette, E. H., Oshiro, I. S., and Cremer, N., .I. N&l. Cancer Inst. 46, II (1971). 9. Bakhanoshvili, M., Wrescher, D. H., and Salzberg, S., Cancer Res. 43, 1289 (1983). 10. Boyum, A., Stand. .I. C/in. Lab. Invest. 97 (Suppl 21), 77 (1968). 11. Dartler, F. J., and Insel, P. A., Exp. Cell Res. 138, 287 (1982). 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). 13. Boer, G. J., Anal. Biochem. 65, 225 (1975). 14. Sassa, S., Zalar, G. L., and Kappas, A., J. C/in. Invest. 61, 499 (1978). 15. Magnussen, C. R., Levine, J. B., Doherty, J. M., Cheesman, J. O., and Tschudy, D. P., BIood 44, 857 (1974).