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Correlation of tumorigenicity with resistance to growth inhibition by cis-hydroxyproline
Printed in Sweden Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/79/140247-06$02.00/O
Experimental
Cell Research 124 (1979) 247-252
CORRELATION OF TUMORIGENICITY WITH RESISTANCE GROWTH INHIBITION BY CZ,S-HYDROXYPROLINE D. VEMBU,’
TO
L. A. LIOTTA,2 M. PARANJPE3 and C. W. BOONE3
‘Laboratory of Molecular Hematology, National Heart, Lung, and Blood Institutes, ZLaboratory of Pathophysiology, National Cancer Institute and Tell Biology Section, National Cancer Institute, NIH, Bethesda, MD 20205, USA
SUMMARY cis-Hydroxyproline, an inhibitor of collagen deposition, was examined for its effect on the growth of a variety of tumorigenic and non-tumorigenic cells. Virally, chemically, and spontaneously transformed murk cell lines were found to be less sensitive to cell spreading and growth inhibition by cis-hydroxyproline (CHP) than were their non-tumorigenic counterparts. The non-tumorigenic lines exhibited a higher rate of collagen accumulation in culture than the tumorigenic cell lines. The rate of collagen accumulation in culture without CHP and the growth inhibition by CHP were directly related. These results suggest that normal but not tumorigenic cells may require synthesis of an extracellular substrate containing collagen to support spreading and growth.
As early as 1944 [l] it was recognized that hydroxyproline, the amino acid characteristically found in collagen, was not directly incorporated into the protein but is synthesized by hydroxylation of peptidyl proline. Hydroxylation of proline is a posttranslational event that occurs intracellularly, close to polyribosomes on the membrane of the endoplasmic reticulum [2]. It is mediated by the specific enzymes prolyl4hydroxylase [3], which catalyzes the formation of 4-hydroxy+proline on the third residue in the triplet (Gly-X-Pro),, where IZ is greater than 2 [4] and by prolyl3-hydroxylase which converts the prolyl residues at X position to 3-hydroxy-L-proline [5]. Hydroxylation occurs on the randomized chain and not on the polypeptide chain in helical configuration [5, 61. Hydroxylation is necessary to stabilize the triple helix of collagen [6], and the helical configuration is
necessary for extracellular secretion of newly synthesized procollagen [7, 81. A number of proline analogs have been developed in an approach to pharmacologically manipulate collagen biosynthesis and deposition [7, 81. These analogs, such as ci+6hydroxyproline, cis-Cfluoroproline, and azetidine-2-carboxylic acid, are incorporated by the cell into the collagen polypeptide chains. cis-4-Hydroxyproline, the analog used here, is incorporated into collagenous peptides and prevents the molecules from assuming a triple helical configuration. For reasons unclear, this in turn leads to increased susceptibility of collagenous peptides to protease degradation and failure of collagen deposition in cultures and tissues [9]. Kao & Prockop first reported that the growth of normal fibroblasts was inhibited by the analog cis-hydroxyproline (CHP) Exp Cell Res 124 (1979)
248
Vembu et al.
while a cultured line of tumor cells was not [lo]. Liotta et al. [ 1I] showed that this analog retarded the spreading and proliferation of adult murine connective tissue cells but not their spontaneously transformed counterpart. In the present study, we have established the generality of the finding that tumor cells are resistant to CHP inhibition as compared to normal cells. The inhibitory effects of CHP were measured over a range of doses and on a variety of normal and neoplastic cells derived mainly from the BALBlc mouse. The tumorigenic potential of these cells has been carefully studied. In addition, we have studied the relationship between the rate of collagen accumulation by these cell lines in culture and their susceptibility to inhibition by CHP.
MATERIALS
AND GROWTH
Growth media All cell lines were mown in Dulbecco-Vogt modified Eagle’s medium supplemented with &eptomycin (100 pg/rnI) and penicillin (100 U/ml) and 10% fetal calf serum (FCS) (complete medium). cis-Hydroxyproline (allo-4-hydroxy-L-proline) was purchased from Calbiothem Co. Stock solutions (1600 pdml) were prepared in complete medium and sterilized by Millipore fdtration (Nalgene Sybron Corp.). This stock solution was kept at -20°C in small aliquots.
Cell lines studied tissue cells (ACT). Murine connective tissue cells were obtained by the subcutaneous iniection of 0.5 % trvnsin and0.5 % collaaenase (Worthington & Co.) as-described previously [ll]: After 45 min, 5 ml of the complete medium was injected into the site and the cells were harvested from the resulting bleb by flushing the medium back and forth. A suspension of cells from three mice was pooled and centrifuged at 2000 rpm for 5 min. The pellet was resuspended in complete medium and the cells that were planted in T-75 flasks became confluent in 2-3 days. TACT-G. ACT cells carried in culture for 15 passages, but not less, produced tumors when injected subcutaneously in syngeneic animals. These tumors were excised, disaaareaated, and slanted in T-75 flasks. The line established was- designated as TACT-G [12]. TACT 2F-6. ACT cells after six passages were
Adult connective
Exp Cell Res 124 (1979)
grown to confluency on I x5x 10 mm plastic plates for 24-48 h and implanted subcutaneously in syngeneic mice. Tumors arose from the cells implanted attached to the plates after 46 weeks and were established in tissue culture as continuous lines. Cells from passage seven were used in this study [ 121. BAD-3T3. This line had developed from inbred BALB/c mice embryo cultures. It is a continuous aneuploid cell line that is sensitive to contact inhibition of cell division [ 131. SV40-3T3. BALB/c mice were inoculated with BALB-3T3 cells transformed by the SV40 virus (provided by Dr George Todaro). The resulting tumor was excised, disaggregated, and established in culture as a
continuouscel,,inelt31
LSQ-3. Skin of BALBjc mice was painted with 0.6 % of 3-methycholanthrene (MCA) in acetone 3 times a week. After 3 months, the resulting squamous cell carcinema of the skin was explanted, disaggregated, and established in culture as a continuous cell line [14]. MCA-6. The MCA-6 cell line was established from explants of fibrosarcomas arising in BALB/c mice 3 months after subcutaneous injection of 0.5 mg of methylcholahthrene in corn oil [ 141. PMT-MET. Highly metastatic librosarcoma cells were derived from a pulmonary metastasis of T-241 tibrosarcoma as described previously [IS]. WZ-38., This diploid tibroblast cell strain derived from female embryonic human lung was obtained from the American Type Culture Collection, Rockville, Md.
Assay for growth inhibition 1X 105 cells suspended in complete medium were planted in 60 mm-Petri dishes. After waiting 4 h for the cells to attach, stock solution of CHP in media was added to achieve a final media concentration of O-200 &ml. The medium was changed every 3rd day. Viable cell counts (trypan blue e&lusion) were determined with a hemacvtometer on the 3rd, 5th and 7th days and the growth ratio compared to controls was calculated. All experiments were performed at least twice, each time in triplicate, and two cell counts were performed on each cell sample. In order to contrast the growth inhibition among the many cell lines, the cell number of the treated group at day 5 was compared with the control cell number. This time period was chosen because all the cell lines studied were in exponential growth by this time.
Assay for the rate of collagen synthesis The rate of collagen synthesis of all cell types was measured by the method of Peterkofsky & Diegelmann [16, 171. Confluent cultures were labeled with [“Clproline for 48 h in the presence of /3-aminoproprionitrile (BAPN) (50 mM) and ascorbic acid (IO mM). Media were harvested from ceil cultures and~dialyzed against 0.05 M acetic acid. The non-dialyzable protein containing accumulated labeled collagen was lyophilized. This protein was digested with purified (Form III Advanced Biofactures) collagenase in the presence of 30 mM N ethyl maleimide. The proportion of ac-
Tumorigenicity
and growth inhibition
cumulated newly synthesized proteins which were collagenous was calculated based on the proportion of collagenase labile proteins [ 16, 171.
RESULTS We first examined the effects of CHP on cell morphology. Adult connective tissue cells sensitive to CHP became rounded and lost their characteristic polygonal shape. The rate of appearance of these changes depended on dose. At low doses (25-50 lug/ml) the majority of cells were rounded by day 3. At higher doses (100-200 pg/ml) initial cell spreading of the freshly plated cells was retarded and most cells were rounded by the end of the first day. As seen by time-lapse cinematography these rounded cells became detached from the substrate. Only 10% of detached cells in the media of CHP-treated cultures were viable as judged by trypan blue exclusion (fig. 1). All cell types studied here which were growth inhibited by CHP, exhibited similar morphologic changes. We next measured the effect of CHP on cell proliferation. All cell types tested were plated at the same density and allowed to attach for 4 h prior to exposure to CHP. Inhibition of cell proliferation was most pronounced during exponential growth. The results of a typical experiment are shown in fig. 2, where the normal connective tissue cells were inhibited by low concentrations of CHP, but their spontaneously transformed counterparts were not. A number of different cell lines were tested for sensitivity to CHP at different concentrations. Tumorigenic cells were consistently less sensitive than the normal cells (table 1). The degree of inhibition ranged from 0.0% to 93 % compared to the control. The greatest difference between the tumorigenic and the non-tumorigenic cells was seen at low concentrations of
by cis-hydroxyproline
249
CHP (25 pg/ml). For example, at 25 pg/ml of CHP adult connective tissue cells were inhibited by 89 %. Spontaneous transformation of these cells occurred following passage 15 and their sensitivity to CHP, 25 pg/ml, was altered such that the cells were inhibited to 64% of control. A continuous line of tumor cells produced from this same cell of origin was inhibited only 8% by 25 pg/ml of CHP. Before and after viral transformation 3T3 cells were inhibited to 93 % and 75%, respectively, by 25 pg/ml of CHP. Other established tumor cells studied showed little or no sensitivity to CHP at this same dosage. Non-transformed human WI38 cells, after many passages in culture, were very sensitive to CHP. Their growth was reduced by 90 % at 25 pg/ml of CHP. The sensitivity to CHP was compared to the rate of collagen accumulation in culture for the cell lines shown in table 1, measured in the absence of CHP. Reduction in cell number due to 25 pg/ml of CHP shown in table 1 was plotted against percent of collagenase-sensitive labeled protein accumulated by the cells (fig. 3). The amount of collagen appeared directly related to the sensitivity of the cells to CHP. Tumor cells accumulated less collagen than the nontumorigenic ones and were less sensitive to the analog. On the other hand, the nontumorigenic cells showed the highest rate of collagen accumulation and also the greatest sensitivity to CHP. DISCUSSION Administration of low doses of CHP has previously been shown to reduce collagen deposition in the tissues of several animal models without producing toxicity [ 181. Studies on aorta-derived cells in vitro demonstrated that incorporation of CHP into protein (at concentrations greater than Exp Cell Res 124 (1979)
250
Vembu et al.
Fig. 1. Morphologic changes in cells sensitive to cishydroxyproline. Adult murine connective tissue cells grown for 36 h in tissue culture dishes (a) in the absence of CHP; (b) 50 pg/ml CHP; (c) 100pg/ml CHP; (d) 200 pg/ml CHP. Untreated cells are flattened and
E-rr, Cd Res 124 11979)
spread. At 50 pg/ml most of the cells have begun to round up. At 100pg/ml most of the cells remaining attached to the dish are rounded. At 200 pg/ml all attached cells are rounded. (a, b, d) X 100; (c) x400.
Tumorigenicity and growth inhibition by cis-hydroxyproline
2.51
Fig. 2. Abscissa: time in culture (days); ordinate: cell no. 0, Control; 0, CHP 25 &ml. Example growth inhibitory effects of low dose CHP on (a) adult murine connective tissue cells and (b) their spontaneously transformed counterparts. CHP treatment was begun 4 h after cells had attached (equal plating efficiency for the two cell types). a
b
those used in the present study) did not have any effect on the synthesis and secretion of elastin, whereas no intact procollagen molecules were recovered [19, 201. Thus, at low doses, cis-hydroxyprolinecan have a specific effect on collagen synthesis and secretion in vivo and in vitro. In the present study we have found that sensitivity to CHP is reduced in tumorigenic cells and Table 1. CHP dose (pglml)
directly related to their rate of collagen accumulation, The inhibition of cell proliferation in vitro by cis- hydroxyproline could be due to more than one mechanism. Certain normal cells may partially require an extracellular matrix of collagen for attachment, spreading, and cis-Hydroxysubsequent proliferation. proline blocks the secretion of such a necessary matrix to which cells bind via the transformation-sensitive protein fibronectin. This conclusion is supported by the finding
Growth inhibition Cell type ACT 1.2 early passage” ACT 1.7 late passage” TACT 2F6 spontaneously transformedb TACT G soontaneouslv transformed” 3T3” SV40-3T3” LSQ-3’ MCA-6’ PMT tibrosarcomad WI38 human fibroblasts
2.5 (%I’ 89 86
90.2 85.1
94.7 92.1
64
82
92.2
9:
f:
96.3 94.8
75 8 26 0 90
89.5 7 54 10 95
94 100 66 YE
LI Non-tumorigenic 100% of syngeneic mice (1 x 10’ cells injected, O/l5 mice developed tumors). b 2.1 x lo4 ACT cells passage 6 were implanted attached to plastic plates [ 121; l/10 mice developed a tumor which was disaggregated and placed in culture. c Spontaneously transformed and chemically and virally transformed cells which are tumorigenic in greater than 90% of syngeneic mice after injection of 1X 10’ ceils. d 100% tumorigenic and 100% metastatic in syngeneic mice after injection of 1X lo6 cells. e No growth compared with control is 100% inhibition.
1
2
3
4
09 8 80 I I 6
Fig. 3. Abscissa: collagen synthesis (% of proteins); ordinate: growth inhibition by CHP (% of control). Relationship between collagen synthesis and inhibition by cis-hydroxyproline. The inhibition of growth is plotted as % of control at 5 days in culture with 25 pg CHP/ml. The % collagen synthesized is the % of [“Clproline incorporated and secreted in proteins which are collagenous for cells grown under control conditions. 0, Tumorigenic; 0, non-tumorigenic. Each point is the mean of duplicate experiments with the range less than 10% of the mean. I, PMT metastatic ftbrosarcoma; 2, TACT G; 3, LSQ; 4, MCA; 5, TACT 2F-6; 6, SV40-3T3; 7, 3T3; 8, WI38; 9, ACT 1.7; IO, ACT 1.2. Exp Cdl Res 124 (1979)
252
Vembu et al.
that exogenous addition of a collagen matrix We are grateful to Hynda Kleinman and George R. Martin for many helpful suggestions on the design of to the culture dish can reverse the effects of the experiments. CHP for normal cells [I 11. It was also established in these prior studies that tumor REFERENCES cell collagen synthesis, although lower than 1. Stetten, M R & Schoenheimer, 0 R, J biol them normal cells, was reduced by CHP to the 153(1944) 113. same extent as normal cells. In addition, at 2. Diegelmann, R F, Bernstein, L & Peterkofsky, B, .I biol them 248 (1973) 6514. high doses of CHP it is possible that the 3. Kivirikko, K I & Prockop, D J, Arch biochem presence of defective collagen containing biophys 118 (1967) 611. 4. Miller, R L & Udenfriend, S, Arch biochem biocis- hydroxyproline within the cell could ohvs 139 (1970) 104. have an effect on the metabolism of other 5. Tr&gvason, K, Risteli, J & Kivirikko, K I, Biothem bioohvs res comm 76 (1977) 275. proteins that participate in cell growth such 6. Berg, R A & Prockop, D J,‘Biodhem biophys res as fibronectin. This would explain why commun 52 (1973) 115. 7. Harsch, M, Murphy, L & Rosenbloom, J, FEBS tumorigenic cells are sensitive to CHP at lett 26 (1972) 48. high doses even though they may not re8. Uitto, J, Dehm, P & Prockop, D J, Biochim biophys acta 278 (1972) 601. quire a collagen matrix for growth. It is con9. Kao, W W-Y, Prockop, D J & Berg, R A, J biol ceivable that tumor cells may have altered them. In press. 10. Kao, W W-Y & Prockop, D J, Nature 266 (1977) CHP transport. 63. Transformation of cells causes a number 11. Liotta, L A, Vembu, D, Kleinman, H, Martin, G R & Boone, C, Nature 272 (1978) 622. of changes in cell morphology, growth rate, 12. Boone, C W, Takeichi, N, Eaton, S & Paranjpe, and synthesis of various cell proteins, inM, Science 204 (1979) 177. cluding collagen [21]. Reduced rates of col- 13. Aaronson, S A & Todaro, G, J cell physiol 72 (1968) 141. lagen accumulation by transformed cells in 14. Boone, C W, Lundberg, E, Orme, T & Gilleli, R, J natl cancer inst (1973). culture could be due to decreased synthesis, 15. Liotta, L A, Vembu,’ D, Saini, R K & Boone, C, or increased degradation, or both mechCancer res 38 (1978) 1231. anisms. The finding that tumorigenic cells 16. Peterkofsky, B, Arch biochem biophys 152 (1972) 318. are less inhibited by CHP and accumulate 17. Peterkofsky, B & Diegelmann, R, Biochemistry 10 (1971) 988. less collagen compared to their normal 18. Daly, J M, Steiger, E, Prockop, D J & Dudrick, S counterparts appears to be a general finding J. J sum res 14 (1973) 551. with regard to at least BALB/c murine cells. 19. Uitto, j, Hoffman, H P & Prockop, D J, Arch biochem biophys 173 (1976) 187. Tumor cells which are anchorage-independ- 20. Rosenbloom, J & Cywinski, A, Biochem biophys res commun 69 (1976) 613. ent may not require an extracellular sub21. Hata, R & Peterkofsky, B, Proc natl acad sci US strate of proteins for attachment and 74 (1977) 2933. growth. This necessary substrate may conReceived February 13, 1979 tain collagen and other proteins such as Revised version received June 11, 1979 Accepted June 18, 1979 fibronectin.
Exp Cell Res 124 (1979)
Experimental
Cell Research 124 (1979) 247-252
CORRELATION OF TUMORIGENICITY WITH RESISTANCE GROWTH INHIBITION BY CZ,S-HYDROXYPROLINE D. VEMBU,’
TO
L. A. LIOTTA,2 M. PARANJPE3 and C. W. BOONE3
‘Laboratory of Molecular Hematology, National Heart, Lung, and Blood Institutes, ZLaboratory of Pathophysiology, National Cancer Institute and Tell Biology Section, National Cancer Institute, NIH, Bethesda, MD 20205, USA
SUMMARY cis-Hydroxyproline, an inhibitor of collagen deposition, was examined for its effect on the growth of a variety of tumorigenic and non-tumorigenic cells. Virally, chemically, and spontaneously transformed murk cell lines were found to be less sensitive to cell spreading and growth inhibition by cis-hydroxyproline (CHP) than were their non-tumorigenic counterparts. The non-tumorigenic lines exhibited a higher rate of collagen accumulation in culture than the tumorigenic cell lines. The rate of collagen accumulation in culture without CHP and the growth inhibition by CHP were directly related. These results suggest that normal but not tumorigenic cells may require synthesis of an extracellular substrate containing collagen to support spreading and growth.
As early as 1944 [l] it was recognized that hydroxyproline, the amino acid characteristically found in collagen, was not directly incorporated into the protein but is synthesized by hydroxylation of peptidyl proline. Hydroxylation of proline is a posttranslational event that occurs intracellularly, close to polyribosomes on the membrane of the endoplasmic reticulum [2]. It is mediated by the specific enzymes prolyl4hydroxylase [3], which catalyzes the formation of 4-hydroxy+proline on the third residue in the triplet (Gly-X-Pro),, where IZ is greater than 2 [4] and by prolyl3-hydroxylase which converts the prolyl residues at X position to 3-hydroxy-L-proline [5]. Hydroxylation occurs on the randomized chain and not on the polypeptide chain in helical configuration [5, 61. Hydroxylation is necessary to stabilize the triple helix of collagen [6], and the helical configuration is
necessary for extracellular secretion of newly synthesized procollagen [7, 81. A number of proline analogs have been developed in an approach to pharmacologically manipulate collagen biosynthesis and deposition [7, 81. These analogs, such as ci+6hydroxyproline, cis-Cfluoroproline, and azetidine-2-carboxylic acid, are incorporated by the cell into the collagen polypeptide chains. cis-4-Hydroxyproline, the analog used here, is incorporated into collagenous peptides and prevents the molecules from assuming a triple helical configuration. For reasons unclear, this in turn leads to increased susceptibility of collagenous peptides to protease degradation and failure of collagen deposition in cultures and tissues [9]. Kao & Prockop first reported that the growth of normal fibroblasts was inhibited by the analog cis-hydroxyproline (CHP) Exp Cell Res 124 (1979)
248
Vembu et al.
while a cultured line of tumor cells was not [lo]. Liotta et al. [ 1I] showed that this analog retarded the spreading and proliferation of adult murine connective tissue cells but not their spontaneously transformed counterpart. In the present study, we have established the generality of the finding that tumor cells are resistant to CHP inhibition as compared to normal cells. The inhibitory effects of CHP were measured over a range of doses and on a variety of normal and neoplastic cells derived mainly from the BALBlc mouse. The tumorigenic potential of these cells has been carefully studied. In addition, we have studied the relationship between the rate of collagen accumulation by these cell lines in culture and their susceptibility to inhibition by CHP.
MATERIALS
AND GROWTH
Growth media All cell lines were mown in Dulbecco-Vogt modified Eagle’s medium supplemented with &eptomycin (100 pg/rnI) and penicillin (100 U/ml) and 10% fetal calf serum (FCS) (complete medium). cis-Hydroxyproline (allo-4-hydroxy-L-proline) was purchased from Calbiothem Co. Stock solutions (1600 pdml) were prepared in complete medium and sterilized by Millipore fdtration (Nalgene Sybron Corp.). This stock solution was kept at -20°C in small aliquots.
Cell lines studied tissue cells (ACT). Murine connective tissue cells were obtained by the subcutaneous iniection of 0.5 % trvnsin and0.5 % collaaenase (Worthington & Co.) as-described previously [ll]: After 45 min, 5 ml of the complete medium was injected into the site and the cells were harvested from the resulting bleb by flushing the medium back and forth. A suspension of cells from three mice was pooled and centrifuged at 2000 rpm for 5 min. The pellet was resuspended in complete medium and the cells that were planted in T-75 flasks became confluent in 2-3 days. TACT-G. ACT cells carried in culture for 15 passages, but not less, produced tumors when injected subcutaneously in syngeneic animals. These tumors were excised, disaaareaated, and slanted in T-75 flasks. The line established was- designated as TACT-G [12]. TACT 2F-6. ACT cells after six passages were
Adult connective
Exp Cell Res 124 (1979)
grown to confluency on I x5x 10 mm plastic plates for 24-48 h and implanted subcutaneously in syngeneic mice. Tumors arose from the cells implanted attached to the plates after 46 weeks and were established in tissue culture as continuous lines. Cells from passage seven were used in this study [ 121. BAD-3T3. This line had developed from inbred BALB/c mice embryo cultures. It is a continuous aneuploid cell line that is sensitive to contact inhibition of cell division [ 131. SV40-3T3. BALB/c mice were inoculated with BALB-3T3 cells transformed by the SV40 virus (provided by Dr George Todaro). The resulting tumor was excised, disaggregated, and established in culture as a
continuouscel,,inelt31
LSQ-3. Skin of BALBjc mice was painted with 0.6 % of 3-methycholanthrene (MCA) in acetone 3 times a week. After 3 months, the resulting squamous cell carcinema of the skin was explanted, disaggregated, and established in culture as a continuous cell line [14]. MCA-6. The MCA-6 cell line was established from explants of fibrosarcomas arising in BALB/c mice 3 months after subcutaneous injection of 0.5 mg of methylcholahthrene in corn oil [ 141. PMT-MET. Highly metastatic librosarcoma cells were derived from a pulmonary metastasis of T-241 tibrosarcoma as described previously [IS]. WZ-38., This diploid tibroblast cell strain derived from female embryonic human lung was obtained from the American Type Culture Collection, Rockville, Md.
Assay for growth inhibition 1X 105 cells suspended in complete medium were planted in 60 mm-Petri dishes. After waiting 4 h for the cells to attach, stock solution of CHP in media was added to achieve a final media concentration of O-200 &ml. The medium was changed every 3rd day. Viable cell counts (trypan blue e&lusion) were determined with a hemacvtometer on the 3rd, 5th and 7th days and the growth ratio compared to controls was calculated. All experiments were performed at least twice, each time in triplicate, and two cell counts were performed on each cell sample. In order to contrast the growth inhibition among the many cell lines, the cell number of the treated group at day 5 was compared with the control cell number. This time period was chosen because all the cell lines studied were in exponential growth by this time.
Assay for the rate of collagen synthesis The rate of collagen synthesis of all cell types was measured by the method of Peterkofsky & Diegelmann [16, 171. Confluent cultures were labeled with [“Clproline for 48 h in the presence of /3-aminoproprionitrile (BAPN) (50 mM) and ascorbic acid (IO mM). Media were harvested from ceil cultures and~dialyzed against 0.05 M acetic acid. The non-dialyzable protein containing accumulated labeled collagen was lyophilized. This protein was digested with purified (Form III Advanced Biofactures) collagenase in the presence of 30 mM N ethyl maleimide. The proportion of ac-
Tumorigenicity
and growth inhibition
cumulated newly synthesized proteins which were collagenous was calculated based on the proportion of collagenase labile proteins [ 16, 171.
RESULTS We first examined the effects of CHP on cell morphology. Adult connective tissue cells sensitive to CHP became rounded and lost their characteristic polygonal shape. The rate of appearance of these changes depended on dose. At low doses (25-50 lug/ml) the majority of cells were rounded by day 3. At higher doses (100-200 pg/ml) initial cell spreading of the freshly plated cells was retarded and most cells were rounded by the end of the first day. As seen by time-lapse cinematography these rounded cells became detached from the substrate. Only 10% of detached cells in the media of CHP-treated cultures were viable as judged by trypan blue exclusion (fig. 1). All cell types studied here which were growth inhibited by CHP, exhibited similar morphologic changes. We next measured the effect of CHP on cell proliferation. All cell types tested were plated at the same density and allowed to attach for 4 h prior to exposure to CHP. Inhibition of cell proliferation was most pronounced during exponential growth. The results of a typical experiment are shown in fig. 2, where the normal connective tissue cells were inhibited by low concentrations of CHP, but their spontaneously transformed counterparts were not. A number of different cell lines were tested for sensitivity to CHP at different concentrations. Tumorigenic cells were consistently less sensitive than the normal cells (table 1). The degree of inhibition ranged from 0.0% to 93 % compared to the control. The greatest difference between the tumorigenic and the non-tumorigenic cells was seen at low concentrations of
by cis-hydroxyproline
249
CHP (25 pg/ml). For example, at 25 pg/ml of CHP adult connective tissue cells were inhibited by 89 %. Spontaneous transformation of these cells occurred following passage 15 and their sensitivity to CHP, 25 pg/ml, was altered such that the cells were inhibited to 64% of control. A continuous line of tumor cells produced from this same cell of origin was inhibited only 8% by 25 pg/ml of CHP. Before and after viral transformation 3T3 cells were inhibited to 93 % and 75%, respectively, by 25 pg/ml of CHP. Other established tumor cells studied showed little or no sensitivity to CHP at this same dosage. Non-transformed human WI38 cells, after many passages in culture, were very sensitive to CHP. Their growth was reduced by 90 % at 25 pg/ml of CHP. The sensitivity to CHP was compared to the rate of collagen accumulation in culture for the cell lines shown in table 1, measured in the absence of CHP. Reduction in cell number due to 25 pg/ml of CHP shown in table 1 was plotted against percent of collagenase-sensitive labeled protein accumulated by the cells (fig. 3). The amount of collagen appeared directly related to the sensitivity of the cells to CHP. Tumor cells accumulated less collagen than the nontumorigenic ones and were less sensitive to the analog. On the other hand, the nontumorigenic cells showed the highest rate of collagen accumulation and also the greatest sensitivity to CHP. DISCUSSION Administration of low doses of CHP has previously been shown to reduce collagen deposition in the tissues of several animal models without producing toxicity [ 181. Studies on aorta-derived cells in vitro demonstrated that incorporation of CHP into protein (at concentrations greater than Exp Cell Res 124 (1979)
250
Vembu et al.
Fig. 1. Morphologic changes in cells sensitive to cishydroxyproline. Adult murine connective tissue cells grown for 36 h in tissue culture dishes (a) in the absence of CHP; (b) 50 pg/ml CHP; (c) 100pg/ml CHP; (d) 200 pg/ml CHP. Untreated cells are flattened and
E-rr, Cd Res 124 11979)
spread. At 50 pg/ml most of the cells have begun to round up. At 100pg/ml most of the cells remaining attached to the dish are rounded. At 200 pg/ml all attached cells are rounded. (a, b, d) X 100; (c) x400.
Tumorigenicity and growth inhibition by cis-hydroxyproline
2.51
Fig. 2. Abscissa: time in culture (days); ordinate: cell no. 0, Control; 0, CHP 25 &ml. Example growth inhibitory effects of low dose CHP on (a) adult murine connective tissue cells and (b) their spontaneously transformed counterparts. CHP treatment was begun 4 h after cells had attached (equal plating efficiency for the two cell types). a
b
those used in the present study) did not have any effect on the synthesis and secretion of elastin, whereas no intact procollagen molecules were recovered [19, 201. Thus, at low doses, cis-hydroxyprolinecan have a specific effect on collagen synthesis and secretion in vivo and in vitro. In the present study we have found that sensitivity to CHP is reduced in tumorigenic cells and Table 1. CHP dose (pglml)
directly related to their rate of collagen accumulation, The inhibition of cell proliferation in vitro by cis- hydroxyproline could be due to more than one mechanism. Certain normal cells may partially require an extracellular matrix of collagen for attachment, spreading, and cis-Hydroxysubsequent proliferation. proline blocks the secretion of such a necessary matrix to which cells bind via the transformation-sensitive protein fibronectin. This conclusion is supported by the finding
Growth inhibition Cell type ACT 1.2 early passage” ACT 1.7 late passage” TACT 2F6 spontaneously transformedb TACT G soontaneouslv transformed” 3T3” SV40-3T3” LSQ-3’ MCA-6’ PMT tibrosarcomad WI38 human fibroblasts
2.5 (%I’ 89 86
90.2 85.1
94.7 92.1
64
82
92.2
9:
f:
96.3 94.8
75 8 26 0 90
89.5 7 54 10 95
94 100 66 YE
LI Non-tumorigenic 100% of syngeneic mice (1 x 10’ cells injected, O/l5 mice developed tumors). b 2.1 x lo4 ACT cells passage 6 were implanted attached to plastic plates [ 121; l/10 mice developed a tumor which was disaggregated and placed in culture. c Spontaneously transformed and chemically and virally transformed cells which are tumorigenic in greater than 90% of syngeneic mice after injection of 1X 10’ ceils. d 100% tumorigenic and 100% metastatic in syngeneic mice after injection of 1X lo6 cells. e No growth compared with control is 100% inhibition.
1
2
3
4
09 8 80 I I 6
Fig. 3. Abscissa: collagen synthesis (% of proteins); ordinate: growth inhibition by CHP (% of control). Relationship between collagen synthesis and inhibition by cis-hydroxyproline. The inhibition of growth is plotted as % of control at 5 days in culture with 25 pg CHP/ml. The % collagen synthesized is the % of [“Clproline incorporated and secreted in proteins which are collagenous for cells grown under control conditions. 0, Tumorigenic; 0, non-tumorigenic. Each point is the mean of duplicate experiments with the range less than 10% of the mean. I, PMT metastatic ftbrosarcoma; 2, TACT G; 3, LSQ; 4, MCA; 5, TACT 2F-6; 6, SV40-3T3; 7, 3T3; 8, WI38; 9, ACT 1.7; IO, ACT 1.2. Exp Cdl Res 124 (1979)
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that exogenous addition of a collagen matrix We are grateful to Hynda Kleinman and George R. Martin for many helpful suggestions on the design of to the culture dish can reverse the effects of the experiments. CHP for normal cells [I 11. It was also established in these prior studies that tumor REFERENCES cell collagen synthesis, although lower than 1. Stetten, M R & Schoenheimer, 0 R, J biol them normal cells, was reduced by CHP to the 153(1944) 113. same extent as normal cells. In addition, at 2. Diegelmann, R F, Bernstein, L & Peterkofsky, B, .I biol them 248 (1973) 6514. high doses of CHP it is possible that the 3. Kivirikko, K I & Prockop, D J, Arch biochem presence of defective collagen containing biophys 118 (1967) 611. 4. Miller, R L & Udenfriend, S, Arch biochem biocis- hydroxyproline within the cell could ohvs 139 (1970) 104. have an effect on the metabolism of other 5. Tr&gvason, K, Risteli, J & Kivirikko, K I, Biothem bioohvs res comm 76 (1977) 275. proteins that participate in cell growth such 6. Berg, R A & Prockop, D J,‘Biodhem biophys res as fibronectin. This would explain why commun 52 (1973) 115. 7. Harsch, M, Murphy, L & Rosenbloom, J, FEBS tumorigenic cells are sensitive to CHP at lett 26 (1972) 48. high doses even though they may not re8. Uitto, J, Dehm, P & Prockop, D J, Biochim biophys acta 278 (1972) 601. quire a collagen matrix for growth. It is con9. Kao, W W-Y, Prockop, D J & Berg, R A, J biol ceivable that tumor cells may have altered them. In press. 10. Kao, W W-Y & Prockop, D J, Nature 266 (1977) CHP transport. 63. Transformation of cells causes a number 11. Liotta, L A, Vembu, D, Kleinman, H, Martin, G R & Boone, C, Nature 272 (1978) 622. of changes in cell morphology, growth rate, 12. Boone, C W, Takeichi, N, Eaton, S & Paranjpe, and synthesis of various cell proteins, inM, Science 204 (1979) 177. cluding collagen [21]. Reduced rates of col- 13. Aaronson, S A & Todaro, G, J cell physiol 72 (1968) 141. lagen accumulation by transformed cells in 14. Boone, C W, Lundberg, E, Orme, T & Gilleli, R, J natl cancer inst (1973). culture could be due to decreased synthesis, 15. Liotta, L A, Vembu,’ D, Saini, R K & Boone, C, or increased degradation, or both mechCancer res 38 (1978) 1231. anisms. The finding that tumorigenic cells 16. Peterkofsky, B, Arch biochem biophys 152 (1972) 318. are less inhibited by CHP and accumulate 17. Peterkofsky, B & Diegelmann, R, Biochemistry 10 (1971) 988. less collagen compared to their normal 18. Daly, J M, Steiger, E, Prockop, D J & Dudrick, S counterparts appears to be a general finding J. J sum res 14 (1973) 551. with regard to at least BALB/c murine cells. 19. Uitto, j, Hoffman, H P & Prockop, D J, Arch biochem biophys 173 (1976) 187. Tumor cells which are anchorage-independ- 20. Rosenbloom, J & Cywinski, A, Biochem biophys res commun 69 (1976) 613. ent may not require an extracellular sub21. Hata, R & Peterkofsky, B, Proc natl acad sci US strate of proteins for attachment and 74 (1977) 2933. growth. This necessary substrate may conReceived February 13, 1979 tain collagen and other proteins such as Revised version received June 11, 1979 Accepted June 18, 1979 fibronectin.
Exp Cell Res 124 (1979)