weekly application of a promoting agent such as croton oil which alone is noncarcinogenic

Transcription

1 INHIBITION OF MOUSE SKIN TUMORIGENESIS BY ACTINOMYCIN D BY HARRY V. GELBOIN, MICHAEL KLEIN, AND RICHARD R. BATES CARCINOGENESIS STUDIES BRANCH, NATIONAL CANCER INSTITUTE, BETHESDA, MARYLAND Communicated by Robert J. Huebner, April 2, 1965 The studies of Berenblum, Shubik, and others1 have shown that a single small dose of DMBA topically applied to mouse skin "initiates" the tumorigenic process. The "initiated" tissue foci may then be "promoted" to the visible tumor stage by weekly application of a promoting agent such as croton oil which alone is noncarcinogenic or weakly so. The "initiation" stage is irreversible since the onset of promotion can be delayed for long periods without diminution of tumor yield. Since "initiation" does not require the continuous application of carcinogen, and the single dose of carcinogen rapidly disappears from the skin, the system appears particularly useful for the biochemical analyses of the early events in chemical carcinogenesis. In this system, the susceptible tissue is also observable grossly and can be treated directly with the carcinogen as well as with other agents of known biological activity. The ability to apply agents directly minimizes systemic dilution and metabolism of the active agent. This system has been used to study the effects of a variety of chemical agents on chemical carcinogenesis. Thus, several workers2 have studied the effects of prolonged treatment with SH inhibitors on various stages of the process of skin tumorigenesis. In a previous report,3 we found that actinomycin D, an inhibitor of DNA-dependent RNA synthesist inhibited skin tumorigenesis when applied on the same day as the carcinogen. In -that study, the skin tumors were induced by small doses of DIMBA followed by weekly treatment with croton oil. An observation which may be related to our results is that of Burdette5 who has shown an inhibition by actinomycin D of both the lethal mutation frequency and the appearance of melanotic atypical growth induced by radiation in Drosophila melanogaster. In this study we investigated the effects of actinomycin D, applied at various intervals before and after DMBA, on subsequent tumor formation. This technique enabled us to examine the time required for the "initiation" process to be completed. In addition we also report on the effect of actinomycin D on skin tumorigenesis induced by large single doses of DMBA with no subsequent croton oil treatment and on urethan (ethyl carbamate)-initiated, croton oil-promoted skin tumorigenesis. Methods.-Groups of male mice of the NIH general purpose Swiss strain were treated as follows: The hair was removed from the back of each mouse with electric clippers one day before the start of the experiments. The DMBA and the inhibitor, each in 0.2 ml of acetone, wereapplied topically to the entire back. Concentration of carcinogen and inhibitor and the time of application are indicated in the legends to each table. In each experiment except that of Table 2A, the mice were treated topically once weekly with 0.2 ml of 1.0% croton oil in acetone. The mice were examined weekly for grossly visible papillomas. "Effective totals" shown in each table indicate the number of mice alive in the group at the time the first tumor appeared. "Total number of skin tumors" represents tumors persisting for at least two weeks and measuring 1 mm or more in diameter. Disappearance of DMBA-H3 from mouse skin: Actinomycin D, puromycin, and DMBA were applied according to the schedule and dosage described in Table 1, experiment 2. The experiment was performed in a manner identical to the biologic assay experiments. Twelve /hg DMBA-H3 (S.A. 584 mc/mm) were applied in 0.2 ml acetone. At the indicated time, full thickness skin was 1353

2 1354 PATHOLOGY: GELBOIN, KLEIN, AND BATES PROC. N. A. S. removed from the back of each mouse and extracted successively with 10 ml cold benzene, 10 ml hot benzene (700C, 15 min), and 10 ml hot ethanol (700C, 15 min). This treatment removed over 99% of the solvent extractable radioactivity in the skin. Aliquots of the combined fractions were measured for radioactivity in a Packard liquid scintillation spectrometer. Each point represents an average of 4 animals. Measurement of uridine-h' and leucine-h3 incorporation: The actinomycin, acetone, and puromycin were given at the times and doses described in Table 1, experiment 2. One hour after the last application of the inhibitor or acetone, 85,uC of uridine-h3 (S.A C/mM) in 0.2 ml of acetone were applied topically to the back of-each mouse. One hour thereafter, full thickness skins were removed, frozen in liquid N2, the fat and panniculus carnosus scraped away, and the skins homogenized by the procedure of Somerville and Heidelberger.5 The proteins were precipitated and washed by a modification of the method of Siekevitz,7 except that cold TCA was substituted for hot TCA. Three mg of the dried residue were dissolved in 2 ml of hyamine by sonication for 1 hr, 10 ml of a phosphor solution were added, and the radioactivity was measured in a Packard liquid scintillation spectrometer.8 After purification as described above, duplicate samples were further treated with TCA at 900C for 30 min, further purified as in the Siekevitz procedure, and the radioactivity was measured. The radioactivity shown in Table 4 for the uridine incorporation represents the radioactivity in cold TCA-insoluble material which is released by hot TCA treatment. The experiments measuring DIleucine-H3 incorporation followed the same protocol described for Table 1, experiment 1. One hour after the last application of carcinogen or inhibitor, 86,C of DL-leucine-H3 (S.A mc/mm) in 0.2 ml H20 were injected intraperitoneally. Each mouse was killed 1 hr later. The skins were removed as described above, the proteins were purified by the procedure of Siekevitz,7 and the radioactivity was determined as described above. Results.-Table 1 shows the effect of a single application of DMBA, followed by weekly croton oil application, on papilloma formation in mouse skin. For the carcinogen controls (expt. 1), 20 of 32 mice developed a total of 89 tumors, an average of 2.8 tumors per mouse. In experiments 2 and 3 the DMBA controls TABLE 1 EFFECT OF ACTINOMYCIN D AND PUROMYCIN ON DMBA-CROTON OIL SKIN TUMORIGENESIS Avg. Mice tumors Treatment Effective with Total per % DMBA Expt. Group (compound in Mg) total tumors tumors mouse control 1 1 DMBA (12) DMBA (12) + Act D (84) DMBA (12) + Puromycin (1328) 2 1 DMBA (12) DMBA (12) + Act D (98) DMBA (12) + Puromycin (1666) 3 1 DMBA (20) DMBA (20) + Act D (15) In expt. 1 the DMBA was applied in 0.2 ml of acetone to groups 1 and 2 at 8 hr and group 3 at 6 hr. Fourteen Ag actinomycin D in 0.2 ml of acetone were applied at 0, 3, 6, 10, 13, and 16 hr. Group 3 received mg of puromycin (0.166,g in 0.2 ml of acetone at 0, l1/2, 3, 4'/2, 71/2, 9, 111/2, and 13 hr). Groups not receiving inhibitor or carcinogen received acetone at corresponding times. In expt. 2 DMBA was given to groups 1 and 2 at 7 hr and group 3 at 6 hr. Actinomycin was given (14 Ag/O.2 ml) at 0, 3, 6, 8, 11, 14, and 17 hr, and a total of mg of puromycin were given in 10 doses at 0, 11/2, 3, 41/2, 51/2, 71/2, 9, 11'!2, 13, and 141/2 hr. In expt. 3, DMBA was applied at 2 hr and actinomycin D (5 Mg/0.2 ml) at 0, 5, and 8 hr. All groups received croton oil once weekly beginning one week after the start of the experiment. Expts. 1, 2, and 3 were terminated at the end of 22, 24, and 17 weeks, respectively. developed an average of 2.9 and 4.1 tumors per mouse. In three experiments, when actinomycin D was applied on the same day as DMBA, the average number of tumors per mouse was reduced to 0.6, 0.9, and 1.5, respectively, which represents a 79, 69, and 65 per cent inhibition of tumor formation. The application of relatively high levels of puromycin had little effect on tumor formation. The results

3 VOL. 53, 1965 PATHOLOGY: GELBOIN, KLEIN, AND BATES 1355 with puromycin are not interpretable in terms of a dependence of "initiation" on protein synthesis since we failed to find inhibition of leucine-h3 incorporation in the skin of puromycin-treated mice. Control groups of equal numbers of mice given no DMBA and either acetone, actinomycin D, or puromycin alone developed two or fewer tumors in the entire group. Inhibition by actinomycin D of skin tumorigenesis induced by a single large dose of DMBA (Table 2A): A single application of 200,ug of DMBA with no subsequent croton oil treatment resulted in the formation of 80 tumors in 29 mice of 38 treated, TABLE 2 ACTINOMYCIN D INHIBITION OF SKIN TUMORIGENESIS BY DMBA AND URETHAN Avg. Mice tumors % Effective with Total per Carcinogen Expt. Group Treatment total tumors tumors mouse control A* 1 DMBA DMBA + Act D (98 jsg) DMBA + Act D (168,ug) Bt 1 Buffer only (IP) Urethan (IP) Urethan (IP) + Act D (IP) Urethan (IP) + Act D (topically) * DMBA with no croton oil treatment. t Urethan with croton oil treatment. In expt. A group 2 was given a total of 98 pg of actinomycin D in doses of 14 pg/0.2 ml of acetone at 0, 3, 6. 8, 11, 14, and 17 hr. Acetone was applied at 21, 24, 27, 31, and 35 hr. Group 3 received a total of 168 pg of actinomycin D in doses of 14 pg/0.2 ml of acetone at 0, 3, 6, 8, 11, 14, 17, 21, 24, 27, 31, ani 35 hr. Group 1 received acetone on the same schedule as group 3. All animals received 200 pg of DMBA/0.2 ml acetone at 7 hr. The experiment was terminated at 16 weeks. In expt. B, groups 2, 3, and 4 were given 20 mg of urethan (ethyl carbamate) in 0.1 ml of saline intraperitoneally at 7 hr. Group 1 was given saline only. Group 3 was given 2.2 pug of actinomycin D in 0.15 ml phosphate buffered saline intraperitoneally at 5 and 9 hr. Group 4 was given a total of 98 pg of actinomycin at 0, 3, 6, 8, 11, 14, and 17 hr (14 jg/0.2 ml acetone). All mice received weekly applications of 0.2 ml of 1% croton oil topically to the back. The experiment was terminated at 29 weeks. representing an average of 2.1 tumors per mouse. When a total of 98 /Ag of actinomycin D were applied topically in separate doses within a 20-hr period of DMBA application, 28 tumors appeared in 16 of 32 mice treated, representing an average of 0.9 tumor per mouse. When a total of 168.ig of actinomycin D were given at intervals within a 35-hr period before and after the DMBA, 6 tumors appeared in 4 of 19 mice treated. Thus, 98 and 168,ug of actinomycin D caused a 57 and 86 per cent inhibition of DMBA-induced tumor formation. The inhibitory effect of actinomycin D, therefore, is not related to, or dependent on, subsequent croton oil application. Actinomycin D inhibition of urethan-initiated skin tumorigenesis: Table 2B shows the inhibitory effect of the topical application of actinomycin D on skin tumorigenesis initiated by the intraperitoneal administration of urethan. In control group 2, 22 tumors appeared in 11 of 38 mice treated with 20 mg of urethan followed by weekly croton oil treatment. When actinomycin D was topically applied to the skin on the same day as the urethan was injected, there was a 59 per cent inhibition of subsequent tumor formation. In this group, 8 tumors appeared in 8 of 33 mice treated. When the actinomycin D was administered intraperitoneally, there was no effect on skin tumor formation. This may be due to the large systemic dilution of the actinomycin D and a consequent failure of the inhibitor to reach the skin in effective concentration. Time of actinomycin D treatment and inhibition of DMBA-croton oil skin tumorigenesis: Table 3 shows the effects of actinomycin D applied at different times be-

4 1356 PATHOLOGY: GELBOIN, KLEIN, AND BATES PROC. N. A. S. TABLE 3 TIME OF ACTINOMYCIN D TREATMENT AND INHIBITION OF DMBA-CROTON OIL SKIN TUMORIGENESIS Time of actinomycin Effective Mice with Total Avg. tumors % DMBA Expt. Group D treatment total tumors tumors per mouse control 1 1 None Day zero None Day Day None Day zero Day Day None Day Day Day Day The hourly schedule of actinomycin D treatment is identical to that of Table 1, expt. 2. Actinomycin was applied on the day indicated. All controls were given acetone in place of actinomycin D. Sixteen,ug of DMBA/0.2 ml acetone were given to all mice on day zero. All mice in expts. 1 and 4 received weekly applications of 0.2 ml of 1% croton oil once a week, beginning one week after the start of the experiment. In expt. 2 the croton oil treatments started 4 weeks after the beginning of the experiment. In expt. 3 the croton oil was started 2 weeks after the DMBA was given. Expts. 1, 2, 3, and 4 were terminated at 21, 22, 23, and 21 weeks, respectively. fore and after a single application of 16,ug of DMBA. Table 3, experiment 1 show that actinomycin D applied on the same day as the DMBA caused a reduction in the average number of tumors per mouse from 2.2 to 0.2, representing an inhibition of 91 per cent. In seven experiments the inhibitory effect of actinomycin D applied on day zero ranged from 65 to 95 per cent. Experiment 2 shows that actinomycin D, applied one day after the DMBA, was equally as effective in inhibiting tumorigenesis as actinomycin D given on day zero. However, when actinomycin D was applied 4 days after DMBA, there was only a 40 per cent inhibition of tumor formation. Table 3 also shows that the application of actinomycin D at 7, 11, and 14 days after DMBA was applied had no inhibitory effect on subsequent tumor formation. These results clearly delineate the time required for the completion of the transformation process which is sensitive to actinomycin D. Thus, at 7 days after DMBA application the process is complete in all tissue foci being transformed. At 4 days after DMBA, the process is complete in most of the foci undergoing transformation, while at one day the process was incomplete in more than 90 per cent of the foci undergoing transformation. Thus, the time required for the actinomycin D sensitive transformation is 1-4 days. When actinomycin D was applied at times later than those cited, there appeared to be a stimulatory effect on skin tumorigenesis. Thus, actinomycin D applied at 32 and 74 days after DMBA increased the number of papillomas formed. This result represents a single experiment, however, and requires confirmation. Actinomycin D applied 7 days before DMBA inhibited skin tumor formation by 30 per cent. In other experiments utilizing actinomycin D-H,3 we found that 20 per cent of applied actinomycin D- H3 remained extractable from whole skin at 5 days after its application. Thus, at 7 days we expect some actinomycin D to be present in the skin and this may be the cause for the slight inhibition observed. -One to two weeks after the application of high levels of actinomycin D (84-98,ug) most of the animals showed moderate to extensive skin damage. Serous fluid

5 VOL. 53, 1965 PATHOLOGY: GELBOIN, KLEIN, AND BATES 1357 was exuded through much of the skin 100 which was covered by brown crusts. A few small ulcers were present. These ' lesions healed within two weeks after their i -.DMBA ONLY appearance. At low levels of actino- X---KDMBA +PUROMYCIN mycin D (15,ug) which were equally in- 5 hibitory, relatively little skin damage was IR observed. The damage observed with the Q \ higher doses raises the question of whethers death of the initiated cells accounts for, the inhibition of tumor formation. Appli cation of high levels of actinomycin D one TIME IN HOURS week week after arao DMBA also gave ulceration FIG. 1.-Disappearance of H3 from skin of vmice treated with DMBA-H3. but did not inhibit tumor formation, suggesting that ulceration per se was not the reason for the inhibition. Furthermore, Berenblum9 reviewed the extensive literature on the effects on carcinogenesis of a large number of toxic agents and reported no correlation between the irritating or destructive action of the agent and its ability to alter the carcinogenic response. Our data, however, do not eliminate cell death as the mechanism of actinomycin D inhibition. If this is the mechanism, then initiation involves a rapid proliferation of cells so that at 4-7 days the number of initiated cells is so great that a high level of actinomycin D has no effect on subsequent tumor yield. Figure 1 shows that the inhibitory effect of actinomycin D is not due to an alteration in the rate of disappearance of DMBA from the skin. At 12 hr after the DMBA-H3 was applied, under conditions identical to those used in the biologic experiments, 95 per cent of the radioactivity had disappeared in all those groups. At 2 hr after the DMBA-H3 was applied, per cent had disappeared in the control and inhibitor-treated mice. Thus, there is no apparent effect of actinomycin D or puromycin on the duration of the DMBA presence in the skin. Effect of actinomycin D and DMBA on uridine-h3 and leucine-h3 incorporation into mouse skin: Table 4 shows the effect of actinomycin D and DMBA on uridine- H3 incorporation into cold TCA-insoluble material releasable by hot TCA. The application of either actinomycin D or DMBA resulted in a considerable inhibition of uridine-h3 incorporation. With the doses of actinomycin and DMBA used, there was no significant enhancement of inhibition when both compounds were administered. Thus, when actinomycin D and DMBA were applied singly or TABLE 4 EFFECT OF ACTINOMYCIN D AND DMBA ON URIDINE-H3 AND LEUCINE-H3 INCORPORATION INTO MOUSE SKIN, ~Uridine-HW ~ Leucine-H3 Cpm/mg % Cpm/mg % Group residue Control residue Control 1 Acetone only Actinomycin D DMBA (18 ;&g) DMBA (200p&g) DMBA (18 jug) + Act D DMBA (200;Ag) + Act D Puromycin

6 1358 PATHOLOGY: GELBOIN, KLEIN, AND BATES PROC. N. A. S. together, uridine-hi incorporation was inhibited by per cent. Puromycin had no effect on uridine-h3 incorporation. In respect to the effect of DMABA on RNA synthesis, our results are similar to those reported by Sinclair and McCarter'0 who showed a D]MBA inhibition of cytidine incorporation in skin slices incubated in vitro. The inhibitory effect of actinomycini D on uridine-h' incorporation in mouse skin is consistent with many reports on the inhibition of DNA-dependent RNA synthesis by actinomycin D. In contrast to the inhibitory effect of actinomycin D and DMBA on uridine incorl)oration, both of these agents stimulated incorporation of leucine-h' into skin protein. Puromycin had no effect. The DMNIBA-induced increase ill leucine-h' incorporation is similar to the reported effect of methylcholanthrene on protein synthesis in liver" and in skin.'2 The stimulation of leucine incorporation by actinomycin D is paradoxical but may be related to the reported stimulatory effect of actinomycin D on intestinal phosphatase activity in mice'3 and the enhancement by actinomycin D of the hydrocortisone-induced synthesis of tryptophan pyrrolase and tyrosine transaminase in rat liver. 14 Discussion. Our studies show that the stage of transformation sensitive to actinomycin is largely complete by 4 days after the carcinogen is administered. This supports the concept of an early stage in skin tumorigenesis which is irreversible and rapid. We should like to suggest one definition of "initiation" to be the early stage of skin tumorigenesis which is sensitive to actinomycin D. Our results demonstrate that the maximum time required for this initiation process is of the order of 4 days. It also is possible that initiation occurs more rapidly but only occurs when a cell is in a particular metabolic state or stage in the mitotic cycle. The fact that actinomycin D applied at day one inhibited tumor formation as effectively as when it was applied at day zero indicates that the process of initiation takes at least one day for completion. Various studies", 1-1 have shown that some of the early biochemical events induced by methyleholanthrene are changes in various components of the geneaction system.24 Thus, methylcholanthrene causes large increases in several microsomal enzyme activities" and it increases the rate of microsomal amino acid incorporation," the incorporation of orotic acid-c'4 into nuclear RNA, and both the amount of nuclear RNA and its messenger RNA content.'6 The MAIC-induced increase in one of the microsomal enzymes, benzpyrene hydroxylase, is observed in many rat tissues and is prevented by either actinomycini D or puromycin.'7 Although M\IC does not produce tumors in adult rat liver and many of the changes revert to normal within several weeks, the activity of MC in one tissue suggests the possibility that similar changes are induced by polycyclic hydrocarbons in tissues susceptible to their carcinogenic action. It thus has been suggested that early events in carcinogenesis may be alterations in the expression of specific genetic information."' 16 Others have proposed a similar hypothesis.'8 Many compounds including carcinogens and hormones'9 induce changes in the gene-action systems of tissues which are both susceptible and not susceptible to carcinogenic transformation by the carcinogen. In tissues not undergoing malignant transformation, the alterations in gene expression may revert back to the normal state. In susceptible tissues, the initial changes may represent the early stages of progressive alterations in the gene-action system which culminate in a pattern of gene activity character-

7 VOL. 53, 1965 PATHOLOGY: GELBOIN, KLEIN, AND BATES 1359 izing the tumor state. Recent reports have suggested altered patterns of messenger RNA synthesis in mouse tumor compared to normal mouse tissues.20 If "initiation" involves an alteration of gene expression, then an agent which blocks this activity would be expected to inhibit the initiation process. The finding that actinomycin D inhibits the initiation of tumorigenesis suggests the necessity for the presence of genetic activity during exposure to the carcinogen for the effectuation of the initiation process. With the levels of actinomycin D used, we do not know whether the less sensitive DNA-directed DNA synthesis is inhibited as effectively as the more sensitive DNA-directed RNA synthesis. Thus, our results do not indicate which activity of the gene may be necessary for initiation, i.e., whether it be DNA-dependent RNA synthesis or DNA-dependent DNA synthesis. DeMaeyer21 has shown that actinomycin D and DMBA are similar in their effects on virus replication in cells grown in vitro. Thus, both agents inhibit the replication of DNA viruses and do not inhibit the replication of an RNA virus. The authors have suggested that DMBA and actinomycin may act at similar cellular sites. Actinomycin D is known to exert its action by binding to DNA, and Brookes and Lawley22 have demonstrated that DMBA is bound to mouse skin DNA. Jensen et al.23 have shown that DMBA depresses H3-thymidine incorporation in mammalian cells. In our studies we observed that both DMBA and actinomycin D showed similar effects on uridine and leucine incorporation, inhibiting the former and enhancing the latter. These data suggest the possibility that DMBA may be inducing these effects by interacting with DNA. Summary.-Actinomycin D inhibits skin tumorigenesis induced by a single small dose of DMBA followed by weekly applications of croton oil. When actinomycin D was applied on the same day as the carcinogen, the inhibition ranged from 65 to 95 per cent. When the inhibitor was applied one day later, it was equally effective. The "initiation" process was much less sensitive to actinomycin D at four days after treatment with carcinogen, and insensitive at seven days. Actinomycin D is also an effective inhibitor of "initiation" of skin tumors by urethan and of the induction of tumors by a single large dose of DMBA. The authors wish to thank Dr. Hans Falk for his encouragement and valuable suggestions. The valuable technical assistance of Mr. David Morgan and Mr. Haywood Waters is gratefully acknowledged. 1 Berenblum, I., and P. Shubik, Brit. J. Cancer, 1, 383 (1947); ibid., 3, 109 (1949); Berenblum, I., and P. Shubik, Brit. Med. Bull., 4, 373 (1947); Clayson, D. B., Chemical Carcinogenesis (Boston: Little, Brown and Co., 1962), p Crabtree, H. G., Cancer Res., 5, 346 (1945); Klein, M., J. Natl. Cancer Inst., 2, 175 (1965); Searle, C. E., and D. L. Woodhouse, Cancer Res., 24, 245 (1964). 3 Gelboin, H. V., and M. Klein, Science, 145, 1321 (1964). 4Reich, E., and I. H. Goldberg, in Progress in Nucleic Acid Research (New York: Academic Press, 1964), vol. 3, p Burdette, W. J., Science, 133, 40 (1961). 6 Somerville, A. R., and C. Heidelberger, Cancer Res., 21, 591 (1961). 7Siekevitz, P., J. Biol. Chem., 195, 549 (1952). 8 Vaughan, M., D. Steinberg, and J. Logan, Science, 126, 446 (1957). 9 Berenblum, I., Arch. Pathol., 38, 233 (1944). 10 Sinclair, N. R., and J. A. McCarter, Nature, 203, 521 (1964). 11 Gelboin, H. V., Biochim. Biophys. Acta, 91, 130 (1964); Gelboin, H. V., and N. R. Blackburn, Cancer Res., 24, 356 (1964).

8 1360 PATHOLOGY: GREEN AND GOLDBERG PROC. N. A. S. 12 Oehlert, W., and B. V. Pein, Beitr. Pathol. Anat. Ailgem. Pathol., 128, 300 (1963). 13 Moog, F., Science, 144, 414 (1964). 14 Garren, L. D., R. R. Howell, G. M. Tomkins, and R. M. Crocco, these PROCEEDINGS, 52, 1121 (1964). 15 Conney, A. H., E. C. Miller, and J. A. Miller, Cancer Res., 16, 450 (1956); Conney, A. H., E. C. Miller, and J. A. Miller, J. Biol. Chem., 228, 753 (1957); Conney, A. H., J. R. Gillette, J. K. Inscoe, E. R. Trams, and H. S. Posner, Science, 130, 1478 (1959). 16 Loeb, L. A., and H. V. Gelboin, Nature, 199, 809 (1963); Loeb, L. A., and H. V. Gelboin, these PROCEEDINGS, 52, 1219 (1964); Hishizawa, T., H.-:tsuka, and H. Terayama, J. Biochem., 56, 97 (1964). 17 Wattenberg, L. W., and J. L. Leong, J. Histochem. Cytochem., 10, 412 (1962); Gelboin, H. V., and N. Blackburn, Cancer Res., 24, 356 (1964). 18 Monod, J., and F. Jacob, in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 26 (1961), p. 389; Pitot, H. C., and C. Heidelberger, Cancer Res., 23, 1694 (1963); Boyland, E., Brit. Med. Bull., 20, 121 (1964). 19 Litwack, G., and D. Kritchevsky, Actions of Hormones on Molecular Processes (New York: John Wiley & Sons, 1964). 20 Kidson, C., and K. S. Kirby, Cancer Res., 24, 1604 (1964); McCarthy, B. J., and B. H. Hoyer, these PROCEEDINGS, 52, 915 (1964). 21 DeMaeyer, E., and J. DeMaeyer-Guignard, Science, 146, 650 (1964). 22 Brookes, P., and P. D. Lawley, Nature, 202,- 781 (1964). 23Jensen, E. V., E. Ford, and C. Huggins, these PROCEEDINGS, 50, 454 (1963). 24 Waddington's term to describe the "whole series of biochemical processes which lead from a gene to the phenotypic character by which it is recognized." SYNTHESIS OF COLLAGEN BY MAMMALIAN CELL LINES OF FIBROBLASTIC AND NONFIBROBLASTIC ORIGIN* BY HOWARD GREEN AND BURTON GOLDBERG DEPARTMENT OF PATHOLOGY, NEW YORK UNIVERSITY SCHOOL OF MEDICINE, NEW YORK Communicated by Michael Heidelberger, April 16, 1965 The differentiated state in mammalian cells involves restriction of the synthesis of specialized proteins to certain classes of cells. The synthesis of collagen is thought to be confined mainly to cells of connective tissues, but the degree to which the production of this protein is suppressed in other cell types has not been examined quantitatively. It is now possible to do so, utilizing homogeneous cell populations of established mammalian lines originating from different cell types. Established mammalian cell lines of fibroblastic origin have already been shown to synthesize collagen in cell culture." 2 In surface cultures some lines synthesize a considerable quantity, most of which precipitates as typical fibrils between the cells2' 3 and can easily be detected by the determination of hydroxyproline in the washed cell layer. Such analysis, however, does not take account of any collagen synthesized by the cultures but which fails to precipitate in the cell layer. A more sensitive and less ambiguous way of expressing the degree to which a cell type is committed to synthesis of collagen is based on analysis of entire cultures (cells plus medium) for incorporation of labeled proline into the hydroxyproline residues of collagen and into the proline residues of all proteins. From the ratio of the two values, and the known abundances of hydroxyproline in collagen and of proline in the average cell protein, one may calculate the ratio of collagen being synthesized