standard concentration of 200/~g/ml human platelet lysate, and the lysate was ... the platelet lysate was subjected to digestion by highly purified trypsin, the ...
IN VITRO 16~8):694-'/05;1980 Allrights reserved9
HUMAN PLATELET LYSATE CONTAINS GROWTH FACTOR ACTIVITIES FOR ESTABLISHED CELL LINES DERIVED FROM VARIOUS TISSUES OF SEVERAL SPECIES CAROLINE T. EASTMENT AND DAVID A. SIRBASKU Department of Biochemistry and Molecular Biology, The University of Texas Medical School, P.O. Box 20708, Houston, Texas 77030 (Received September 24, 1979; accepted January 21, 1980)
SUMMARY Factors have been studied from human platelets that promote the growth of a hormoneresponsive rat mammary adenocarcinoma cell line M T W 9 / P L , the B A L B / c 3T3 mouse embryo fibroblasts, and numerous other established cell lines. A wide variety of the commonly employed cell lines, including lines of human, mouse, monkey, chicken, rat, Chinese hamster, and Syrian hamster origin, were tested for their growth response to a standard concentration of 200/~g/ml human platelet lysate, and the lysate was found to contain mitogenic activity for 24 of the 29 different lines assayed. A comparison was made between the platelet growth activity for the M T W 9 / P L cells and the well characterized platelet mitogen for the B A L B / c 3T3 cells, platelet-derived growth factor ~PDGF). When the platelet lysate was subjected to digestion by highly purified trypsin, the mitogenic activity for the M T W 9 / P L ceils was not affected whereas that for the B A L B / c 3T3 cells was essentially destroyed. Crude P D G F was prepared by heating the human platelet lysates at 100 ~ C for 2 min followed by clarification, dialysis, lyophilization, and reconstitution. This P D G F material had no apparent growth activity for M T W 9 / P L cells, although chromatography of this material on Biogel P-100 revealed a high molecular weight ~approximately 40,000 daltons) activity for the B A L B / c 3T3 cells (presumably P D G F ) and two growth activities for the M T W 9 / P L cells, one high molecular weight activity and a second activity of molecular weight less than 10,000. These studies demonstrated a form of epithelial tumor cell growth activity separable from the 3T3 type P D G F in crude heated extracts. K e y words: platelets; mitogens; epithelial cells; mesenchymal cells. INTRODUCTION
blasts. Kohler and Lipton (2) found that an extract of human platelets, when combined with a Recent observations in the search for growth low level of calf serum, was mitogenic for mouse regulatory substances have shown that one of the 3T3 cells. In addition, partially purified factors most potent mitogen sources in the blood is the from human platelets have been shown to stimuplatelet (1,2). Balk 13) observed the growth of late [3H]thymidine incorporation into serum denormal chicken fibroblasts in chicken plasma to prived human glial cells (4,5). Furthermore, be depressed relative to that in chicken serum and Kepner et al. ~6) have reported the presence of a postulated that the thrombocytes were the source factor derived from platelets that stimulated the of the mitogenic activity in serum. Ross et al. ~1) proliferation of SV40-transformed 3T3 ceils, but and Kohler and Lipton ~2) extended this observa- not normal 3T3 cells. tion and actually demonstrated that platelets conThe role of the platelet growth factor in the contain growth factor. Ross et al. (1) showed that .*he trol of cell growth has been examined. An activity addition to plasma of a factor derived from mon- that stimulates confluent B A L B / c 3T3 cells to key platelets promoted the growth of homologous replicate was identified in extracts of human arterial smooth muscle cells and dermal fibro- platelets that had been heated at 100 ~ C for 2 rain 694
PLATELET-DERIVED GROWTH FACTORS ~7). This activity, which was designated plateletderived growth factor (PDGF~, functions synergistically with platelet-poor plasma to induce D N A synthesis in quiescent, density-inhibited B A L B / c 3T3 cells as shown by Pledger et al. t8). Platelet-derived growth factor functions by inducing these cells to become competent, that is, able to respond to a second factor or factors. These second factors, present in platelet-poor plasma, have been designated the progression factors and induce these cells to progress through the Go/G, boundary, enter S phase, and eventually divide t9). Vogel et al. (10j likewise maintain that density-inhibited SWISS 3T3 cells require both platelet-derived growth factors and plasma constituents for cellular division. They have also shown that these two components of blood serum act in a coordinate fashion to stimulate cell proliferation. Furthermore, it has been shown that multiplication stimulating activity (MSA) and a member of the somatomedin family of growth factors, somatomedin C, are both potent mediators of progression ( 11 ~. The various growth factors contained in platelets have been partially characterized. Studies of P D G F have shown that it is stored in the alpha granules of platelets and released following adherence or aggregation of platelets induced by exposure to substrates such as collagen or thrombin t12~. Other work has revealed that P D G F is a heat stable (100 ~ C for 2 min~ cationic, trypsin labile, mercaptoethanol-sensitive, molecule ~10, 14, 16~. Platelet-derived growth factor has recently been purified to homogeneity (13~ and the molecular weight of this molecule under nonreducing conditions is 38,000 daltons. Another platelet growth factor mitogenic for SV40transformed 3T3 cells, but not normal 3T3 cells, has been identified (6) and is reported to be a thiol-seusitive 73,000-dalton molecule. At least three partially purified multiplication activities for human glial cells have been derived from human platelets (5L Two anionic activities of tool wt 40,000 and less than 10,000 were identified, as well as a more heterogeneous cationic activity of mol wt 26,000 to 33,000. These results indicate the presence in platelets of more than one form of growth factor activity, although it was not excluded that these forms could be the same growth factor, either in multiple aggregation states or associated with various other proteins in the platelet lysate. One other important point is that these fractions were not simultaneously assayed for growth activity with 3T3 fibroblasts, which would
695
have determined whether glial cell growth factor is different from the P D G F already identified. We became interested in the platelet-derived growth activity as a potential source of a hormone-controlled growth factor for mammary tumor cells. Along these lines, we have shown that platelets are a rich source of mitogenic activity for M T W 9 / P L cells i14-16~, as well as the human origin M C F - 7 breast cancer cells (15j. The M T W 9 / P L cell line, established in culture from a parent M T - W 9 A hormone-responsive rat mammary adenocarcinoma (17), requires estrogen, prolactin, and thyroid hormones for optimal tumor formation in host animals but in culture responds only to physiological concentrations of Lthyroxine. Neither estrogen nor prolactin were found to be directly mitogenic in vitro. If the data from the in vivo growth experiments are to be reconciled with the in vitro results, secondary or mediated hormone affects may be important in vivo. It has been suggested that estrogenic as well as other hormones (18-21~ facilitate platelet aggregation and release in vivo and in vitro; thus these hormones could induce the release of mitogenicity from platelets in vivo. Furthermore, it has been shown that several tumor cells induce aggregation of homologous platelets in vitro t22); such aggregation could allow the release of mitogenic activity from the platelets. One of the major questions that the present work asks is: do platelets contain mitogenic activity for a broad range of both normal and transformed established cell lines of various tissue and species origin? Indeed, if the platelet contains more than one mitogenic activity, can one separate any of these from the well known platelet-derived growth factor, PDGF? This report describes the presence of mitogenic activity in platelet extracts for many well known cell lines and further shows a separation between the growth factor for 3T3 fibroblasts (PDGF~ and a mitogen for M T W 9 / P L rat mammary tumor cells.
MATERIALS AND METHODS Cell culture. All cell lines were maintained in serum containing Dulbecco-Vogt's modified Eagle's medium (DME) (Grand Island Biological Company, Grand Island, NY, H-21 preparation) supplemented with 2 m M glutamine and 2.2 g sodium bicarbonate/l. The fetal bovine serum (FBS~, newborn bovine serum (NBS) and horse
696
EASTMENT AND SIRBASKU
serum (Grand Island Biological Company) were used without heat inactivation. All cells were grown at 37 ~ C in a humid atmosphere of 95% air:5% CO2. The M T W 9 / P L rat mammary tumor cells were grown as described before ~17). The conditions have been described for growth for the H-301 hamster kidney tumor cells ~23) and the GH3/C14 rat pituitary tumor ceils (24). The majority of the cell lines were grown in D M E supplemented with 10% (vol/vol) FBS. This included the following lines obtained from the American Type Culture Collection (ATCC, Rockville, MD): The CHO-K1 Chinese hamster cells (25), which were grown in proline supplemented medium; HeLa, a human line of epitheloid carcinoma origin (26); BS-C-1 African Green Monkey kidney cells (27); another African Green Monkey line CV-1P (28); the mouse neuroblastoma line Neuro-2a ~29); a mouse renal adenocarcinoma cell line RAG (30); the TCMK-1 cells, an SV40 virus-transformed mouse kidney line (31); and the S-100 protein secreting rat glial cell tumor line C6 (32). The following cell lines, also obtained from ATCC, were grown in D M E supplemented with 12.5% lvol/vol) horse serum and 2.5% (vol/vol) FBS: the adenocorticotropinsecreting AtT-20 mouse pituitary tumor cells (33); the GH1 cells, an autonomous rat pituitary tumor cell line ~34); and the R2C steroid-producing rat Leydig tumor cells ~35). Other lines also obtained from ATCC were grown in D M E supplemented with 10% (vol/vol) NBS: the Syrian baby hamster kidney cell line BHK-21(C-13) (36); the adult Syrian hamster kidney cell line HaK; the B A L B / c 3T3 clone A31 contact-inhibited mouse embryo fibroblast cell line (37), the Don cell line derived from Chinese hamster lung (38), and the Dede Chinese hamster cell line. Primary chick embryo fibroblasts lGrand Island Biological) were grown in D M E supplemented with 5% Ivol/vol) NBS. Two normal human fibroblast cell lines, Micdoo, derived from a young adult male, and Nubon, derived from a male neonate, were obtained from Dr. S. Gigonatelli, Baylor College of Medicine, Houston and were grown in D M E supplemented with 10% (vol/vol) FBS. The MSA secreting liver tumor cell line C R L 139) obtained from Dr. H. Temin, University of Wisconsin, Madison, was grown in D M E supplemented with 10% (vol/vol) NBS. The SV40 virus-transformed B A L B / c 3T3 cell lines, SV-3T3 clones 20, Q, and H, were obtained from Dr. P. Rigby, Imperial College, London and grown in D M E supple-
mented with 10% (vol/vol) FBS. The human mammary tumor cell line MCF-7 (401 grown in D M E supplemented with 20% (vol/vol) FBS was obtained from Dr. R. Cailleau, M . D . Anderson Research Institute, Houston. The A-195 autonomous hamster kidney tumor line was developed by Dr. D. Sirbasku and grown in D M E with 10% (vol/vol) FBS, as was the Rous sarcoma virustransformed baby hamster kidney line RSVB H K , which was obtained from Dr. J. Roman, Purdue University. The mouse adrenal cell tumor ACTH secreting line OS-3, obtained from Dr. G. Sato, University of California, La Jolla, was grown in 12.5% (vol/vol) horse serum and 2.5% (vol/vol) FBS.
Preparation of human platelet lysate and human serum. Outdated 3- to 5-day-old human platelet concentrates were obtained from the Gulf Coast Regional Blood Bank, Houston, TX. Platelet lysates were prepared as previously described (16). The protein concentration of the human platelet lysate (HPL) was determined spectrophotometrically assuming 1 A 280 nm ---1 m g / m l and was routinely found to be within the range of 3 m g / m l or 15 mg/platelet unit. The amount of residual plasma in the platelet lysate preparation has been estimated to be less than 5% of the total protein by polyacrylamide gel electrophoresis assay. The lysate contains a majority of protein bands separable from those of the corresponding platelet poor plasma. Freshly drawn citrated whole blood was obtained from Community Blood and Plasma, Houston, T X . Serum was prepared as previously described ~16) and the material was then filter sterilized and stored at - 2 0 ~ C until use. Assay for growth activity. The mitogenic activity of the various preparations was determined by their ability to cause an increase in cell number within the designated time period. Cells were inoculated at a density of 3 x 104 or 4 x 104 cells (depending on the cell line) per 35-mm diameter tissue culture dish (Corning Glass Works, Corning, NY) in 2.5 ml of the standard serumcontaining growth medium for the cell line being tested. After 18 to 24 hr at 37 ~ C, to allow maximum cell attachment, the medium was removed and replaced with the test samples in 3.0 ml of serum-free D M E . All experiments included a background control of 3.0 ml of serum-free D M E alone. At the time of medium change, cells from triplicate zero time control plates were harvested by trypsinization and the cell number was determined with a Coulter Counter (Model ZBI). The
PLATELET-DERIVED GROWTH FACTORS cell growth experiments with the M T W 9 / P L cells were harvested after 6 days. The BALB/c 3T3 cells were harvested after 5 days. In those cases where the duration of the growth curve was different, this fact was noted. Heat treatment of platelet lysate. Sterile HPL was diluted to a standard concentration of 2.6 mg/ml with PBS. One- or two-ml aliquots of HPL were transferred to sterile glass ampules, which were then sealed, placed in a boiling water bath for various time periods, and then rapidly cooled on ice. A control sample was held at 4 ~ C for the duration of the heating. pH Treatment of platelet lysate. H u m a n platelet lysate was taken to pH 4.5 by the dropwise addition of 1 N HCI, stirred 30 min at 4 ~ C, returned to neutral pH with 1 N NaOH and centrifuged at 39,000 xg for 20 min to remove the precipitate. The platelet lysate, which was acid extracted in this manner, was tested on the cells at a volume equal to that of the untreated HPL, yielding the desired protein concentration.
Carboxymethyl
Sephadex
chromatography.
Human platelet lysate (80 ml, 2.9 mg/ml), dialyzed against 0.01 M sodium phosphate buffer, pH 7.2, was applied to a 2.5 x 28-cm column of CM-Sephadex C-50 ~Pharmacia Fine Chemicals, Piscataway, N J), equilibrated with the same buffer. The column was eluted with 0.4 M NaCI in the 0.01 M sodium phosphate, pH 7.2. The effluent was analyzed for protein at 280 nm. The total protein recovered was 96% of that loaded; of the recovered protein 70% was nonabsorbed and 30% eluted by 0.4 M NaCl eluted material and these two pools were dialyzed, lyophilized, reconstituted, and tested for growth activity.
RESULTS
Cell line growth in culture medium supplemented with human platelet lysate. In order to determine the species and cell type range of the mitogenic activity in platelet lysate a wide variety of the commonly employed cell lines were tested for their growth response to human platelet lysate. The cell growth in 200 ~g/ml HPL was compared to that observed in D M E alone and in DME containing 5% tvol/vol) human serum, a level previously shown to yield optimum M T W 9 / P L cell growth. The data of Table 1 show that the standard concentration of 200~g/ml H P L (a concentration previously shown to yield optimum M T W 9 / P L cell growthl
697
was mitogenic for 24 of the 29 different cell lines assayed. The lines tested included cells of human, mouse, monkey, chicken, rat, Chinese hamster, and Syrian hamster origin; yet only chicken embryo fibroblasts, BS-C-1 cells, CHO ovary cells, HAK kidney cells, and OS-3 adrenal cells did not respond to HPL, In the majority of the cases where cell growth was observed in HPL, it was at least 50% of that in the 5% human serum. That is, platelets would appear to represent a major source of the mitogens contained in human serum. H u m a n platelet lysate potentiated the growth of cells of epithelial origin such as the MCF-7 human mammary tumor and the M T W 9 / P L rat mammary adenocarcinoma, as well as cells of fibroblastic origins, i.e., BALB/c 3T3 and newborn human fibroblasts. Both normal and transformed cells grew in the presence of HPL, although a proportionally greater response was observed in the SV-3T3 cells as compared to BALB/c 3T3 and in RSV-BHK cells as compared to BHK. The mitogenic activity(s} was found to be equally potent for both hormoneresponsive and autonomous cells of similar origin, as observed in the estrogen-responsive Syrian hamster kidney tumor H-301 and the autonomous variant A-195, as well as the GH~/C,4 estrogenresponsive rat pituitary tumor and its autonomous variant GH,. H u m a n platelet lysate was also shown to be mitogenic for cells known to express a differentiated function including rat C6 glioma cells, the R2C rat Leydig cell tumor, and the neuroblastoma cells. Stability of human platelet lysate at 100 ~ C. It should be noted that in the cases of the BALB/c cells and the human fibroblasts, growth occurs in the absence of added platelet-poor plasma. Since the assays are conducted by first plating the cells in serum-containing medium, followed by removal of the majority of the serum, it would appear that sufficient serum remains to provide the necessary progression factors for growth. From this extensive list of cell lines, we chose five established lines, both normal and transformed, of rodent and human origin for further studies. These lines were: BALB/c 3T3, SV40 virus-transformed 3T3 (clone Q), the Nubon line, the M T W 9 / P L tumor line, and MCF-7 cells. In order to determine whether separate growth factor activities were responsible for the growth of these various lines, the stability at 100 ~ C of the mitogenic activity in the HPL was determined. Under the experimental conditions employed, the majority of the growth activity in the H P L for the
698
EASTMENT AND SIRBASKU TABLE 1 MITOGENIC ACTIVITY OF HUMAN PLATELET LYSATE FOR A VARIETY OF CELL LINES Number of Cell Population Doublings
Species of Origin
Name of Cell Line
5% Human Serum
Serum-Free Medium
200 ~g/ml HPL
Duration of Growth Experiment
Aves
1. Chick embryo fibroblasts
0 0 0
0 0 0
0 0 0
da~ 5.0 6.0 6.0
Hamster
2. A-195: autonomous kidney t u m o r (Syrian)
3.9 3.5 2.7
2.5 2.2 1.5
4.6 3.7 3.4
4.9 5.1 5.0
3. BHK-21{C-13): baby hamster kidney {Syrian)
5.0 4.1
0.5 0
1.5 1.2
4.0 4.0
4. R S V - B H K : R o u s s a r c o m a virus-transformed baby h a m s t e r kidney (Syrian)
5.2 5.0 7.3
0 0 0
3.0 2.1 3.1
2.9 3.0 4.9
5. C H O - K I : ovary {Chinese)
3.8 4.5 3.2
1.2 0.9 1.2
1.6 0.8 2.1
7.0 4.0 3.0
6. Dede: female lung (Chinese)
5.9 4.7 3.8
0 0 0
2.2 2.0 1.1
3.9 4.7 5.1
7. Don: male lung iChinese)
4.1 4.5 5.3
0 0 0
0.4 1.5 0.6
5.1 4.8 3.9
8. H A K : kidney{Syrian)
3.8 2.8 2.7
2.0 2.2 ().6
2.8 2.8 2.1
6.3 5.0 3.0
9. H-301 : hormone responsive kidney t u m o r (Syrian)
6.1 5.2 5.0
1.2 1.9 0.8
6.() 5.0 5.0
6.0 5.8 4.0
10. HeLa: cervical carcinoma
4.3 5.1 3.7
0.6 0.3 0
3.2 4.1 2.7
4.0 4.{) 4.0
l l. MCF-7: m a m m a r y tumor
2.8 3.5 4.1
2.1 1.5 1.9
2.6 2.9 3.5
5.2 5.9 6.0
12. Micdoo: fibroblast iyoung adult)
1.4 1.2
0 0.3
0.7 1.3
4.9 5.0
13. Nubon: fibroblast {neonate)
1.8 1.5 2.3
0 0 0
1.4 1.5 2.2
5.0 5.0 6.O
14. CV-IP: kidney tAfrican Green)
3.8 3. l 2.4
0.3 0 0.7
3.1 2.{) 1.5
4.0 3.7 3.8
15. BS-C-1 : kidney (African Green )
0 0 0
0 0 0
0 0 0
6.0 6.0 6.0
16. AtT-20: pituitary tumor A C T H secreting
5.1 3.8
0 0
2.4 2.4
7.2 5.0
17. B A L B / c 3 T 3 : contact inhibited, nontumorigenic
2.7 2.8 2.2
0 0.l 0
1.2 2.4 1.3
5.0 6.0 5.0
18. SV-3T3, clone 20: SV40transformed B A L B / c 3T3
5.6 6.1 6.0
0 0 0.8
4.5 4.3 4.9
5.9 4.8 4.1
Human
Monkey
Mouse
699
PLATELET-DERIVED GROWTH FACTORS TABLE 1 ~continued) Number of Cell Population Doublings Species of Origin
Name of Cell Line
19. SV-3T3, clone H: SV40transformed BALB/c 3T3 20. OS-3: adrenal cell tumor
21. Nearo-2a: neuroblastoma
22. RAG: renaladenocarcinoma
23. TCMK-I: SV40-transformed kidney Rat
24. C6: glial cell, S-100, protein secreting
25. CRL: liver tumor, MSA secreting 26. GH,: pituitary tumor, autonomous 27. GH,/Cl4: pituitary tumor, hormone responsive 28. MTW9/PL: mammary adenocarcinoma, hormone responsive 29. R2C: Leydig cell testicular tumor, steroid secreting
five cell lines tested was stable to heating in sealed ampules at 100 ~ C for as long as 15 rain. In fact, at no time was the loss of activity much greater than 30% of the control activity observed for any of these lines. By the criteria of heat stability, the mitogenic factor(s) in H P L for the different cell lines could not be separated. Ion-exchange chromatography. Fractionation by ion-exchange chromatography was employed in an attempt to separate the growth factor activities for the different cell lines. Ion-exchange chromatography on carboxymethyl Sephadex C50 of the h u m a n platelet lysate separated a nonabsorbed activity from the bound activity that was eluted with 0.4 M NaCI. T h e mitogenic activity of both of these pools and of the precolumn material were essentially equal (on a microgram
5% Human Serum
Serum-Free Medium
200/xg/ml HPL
Duration of Growth Experiment
6.0 7.0 7.0 2.7 3.9 3.8 5.9 6.8 6.8 6.8 6.2 6.8 7.2 7.6 6.0 5.8 6.3 5.6
0 0 0.8 1.4 2.2 2.6 0.6 0 1.5 1.3 0.9 0 0 1.1 1.2 2.0 0.2
5.9 6.4 6.7 1.8 2.8 3.1 4.7 4.3 5.1 3.7 4.4 3.9 4.4 4.5 4.1 5.7 5.7 6.1
5.8 6.1 6.8 5.0 6.0 6.0 4.0 5.8 4.1 4.8 4.9 5.0 4.2 4.2 3.1 4.1 5.8 4.0
3.7 2.0 2.2 3. I 3.2 2.8 4.2 3.2 5.7 4.3 4.5 3.6 1.5 3.2
1.4 0.4 0.7 0 0.5 0 0.5 0.1 0.9 0.2 0 0 0 0.3
3.9 1.9 2.1 2.4 2.3 1.8 2.3 1.6 3.8 3.1 2.8 2.3 0.8 2.0
8.0 4.0 4.0 6.0 5.0 6.O 6. l 6.0 7.0 4.8 5.8 6.0 4.0 6.0
2.2
per milliliter basis) for all the five lines tested ~Table 2). Here again, no separation of the various activities was achieved, although an important point was established that in crude lysates of platelets, the growth activity for all cell lines tested separates into two forms: one binds to carboxymethyl Sephadex and must be eluted by high salt treatment and a second form, which passes through the column. T h e bypass fraction has been reapplied to a second carboxymethyl Sephadex column under the same conditions as the first and all the activity eluted in the flow-through fractions. These data indicated that the two earboxymethyl Sephadex forms are not in simple equilibrium ~possibly carrier bound and free form?), but that these are stable forms of platelet mitogenic activity.
700
EASTMENT AND SIRBASKU TABLE 2 GROWTH OF VARIOUS CELL LINES IN FRACTIONS OBTAINED FROM CM-SEPHADEX CHROMATOGRAPHY OF HUMAN PLATELET LYSATE
Cell line
Precolumn HPL
Number Cell Doublings
Nonabsorbed
~g/ml BALB/c 3T3
SV-3T3 clone Q
MCF-7
MTW9/PL
Nubon
0 100 50 0 100 50 0 100 50 0 100 5O 0 100 50
Number Cell Doublings
#g/ml 0.5 2.2 1.2 2.7 4.3 4.1 1.3 2.1 4.1 0.3 3.5 2.9 0.4 1.7 1.9
Acidification of human platelet lysate and heat stability of the material. At this point, the work was limited to two cell lines, the BALB/c 3T3 cells and the M T W 9 / P L cells, and the focus of the studies became the evaluation of the presence of more than one growth factor in the platelets. The best characterized platelet growth factor is platelet-derived growth factor ~PDGF) (41~, however, evidence has been presented that suggests the existence of additional platelet growth factors for other cells (5,27L The BALB/c 3T3 cells were chosen since their mitogenic response to P D G F is well established (41~ and M T W 9 / P L cells were employed since it was believed that they may respond to a platelet growth factor different from PDGF. Acidification of crude homogenates is typically an initial step in the preparation of growth factors such as fibroblast growth factor t42). Therefore, the HPL was taken to pH 4.5 with 1 N HCI [see Materials and Methods) and the growth promoting ability of this material compared to that of the control HPL was tested on BALB/c 3T3 cells and M T W 9 / P L cells. While this procedure resulted in the loss of at least 25% of the protein, there was no loss of growth activity for either cell line. When the acidified HPL was subjected to heating at 100~ C in sealed ampules, the stability of the growth activity was essentially the same as that of the untreated H P L for both M T W 9 / P L and BALB/c 3T3 cells.
P-IO0 Chromatography of human platelet lysate and acidified human platelet lysate. In a
Absorbed
Number Cell Doublings
#g/ml 1.3
100
5O
1.9
50
2.9 1.5
100 5O
3.8 3.3
100 5O
4.7 4.5
100 5O
2.7 3.3
100 5O
4.7 4.5
100 50
3.3 2.8
100 50
3.5 3.1
100 5t)
2.7 2.1
100 5O
2.6 2.2
100
further attempt to separate the mitogenic activity for the BALB/c 3T3 cells from that for the M T W 9 / P L cells, chromatography on Biogel P100 was performed. Chromatography of the HPL (Fig. 1, ,4) and the acidified HPL (Fig. 1, B) revealed a broad region of mitogenic activity eluting from the column slightly after human serum albumin in both cases. The apparent molecular weight range for the growth activities for both the BALB/c 3T3 and the M T W 9 / P L was from 30,000 to 70,000 daltons. Again, a cell specific separation of growth activity was not observed.
.4 time course of the trypsin stability of the growth activity(s) in the lysate. A time course of the trypsin stability of the growth activity in the human platelet lysate for the M T W 9 / P L cells, as compared to that for the BALB/c 3T3 cells, was performed. As depicted in Fig. 2, a clear difference was observed between the trypsin stability of the growth activities in the lysate for the two cell lines. While no loss of the M T W 9 / P L growth activity was observed over the 8-hr time period, a loss of 70% of the 3T3 activity was seen.
Titration and P-IO0 chromatography of platelet-derived growth factor. Up to this point all the data, with the exception of the trypsinization results, suggested that the BALB/c 3T3 growth factor and the M T W 9 / P L growth factor were the same. To further explore the problem, a preparation of platelet-derived growth factor (PDGF~ was made according to the method of Antonaides and Scher ~7). A titration of this crude P D G F for the BALB/c 3T3 cells attained a plateau at
701
PLATELET-DERIVED GROWTH FACTORS
50/ag/ml, at which point four cell population doublings were observed (Fig. 3). However, at no concentration from 10 ~g/ml to 200/ag/ml was any M T W 9 / P L cell growth observed with P D G F (Fig. 3}. In fact, the growth was actually less than that observed in serum-free medium, which typi-
cally ranged from 0.5 to 1.0 population doublings for the M T W 9 / P L cells. This latter result suggested the presence of an inhibitor of the growth of the M T W 9 / P L cells in the crude P D G F preparation. Consequently, the P D G F material was chromatographed on Biogel P-100 in an attempt
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13 700
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Fraction Number 13,700
Void 67,000 45,000
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6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
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F I(;. 1. Elution profiles of Biogel P-100 chromatography of h u m a n platelet lysate and acidified h u m a n platelet lysate. Fractions ~4.0 mD were collected, assayed at 280 n m for protein concentration, and 0.3 ml of each fraction was added to 2.7 ml of D M E and assayed for M T W g / P L and B A L B / c 3T3 cell growth, as described in Materials and Methods. T h e elution volume of the following marker proteins are shown above the elution profile: bovine serum albumin ~mol wt, 67,000), ovalbumin (tool wt, 43,000), and ribonuclease A lmol wt, 13,700}. A, H u m a n platelet lysate {6.4 m g / m l , 5 mD was applied to a 1.5 x 88-cm column of Biogel P-100 equilibrated with PBS. B, Lysate treated at pH 4.5 ~Materials and Methods} ~3.4 m g / m l , 6 mD was applied to a 1.5 x 88 column of Biogel P-100 equilibrated with PBS.
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FI6. 2. A time course of the trypsin stability of the growth activity(s) in the human platelet lysate for the MTW9/PL cells and the BALB/c 3T3 cells. Sterile human platelet lysate (4.0 mg) was incubated with 4.0 mg trypsin at 37 ~ C for various time periods after which the reaction was stopped by the addition of 8.0 mg soybean trypsin inhibitor. The samples were brought to 20 ml total volume with DME and tested on the two cell lines, as described in Materials and Methods. Control samples with only soybean trypsin inhibitor added showed no effect on growth of either the MTW9/PL or 3T3 cells. to separate any possible growth inhibitory substance. As seen in Fig. 4, the P D G F material does in fact contain significant mitogenic activity towards the M T W 9 / P L cells. A region of activity for both the B A L B / c 3T3 cells and the M T W 9 / P L cells eluted from the column in the mol wt range of 75,000 to 40,000, and, in addition, a second region of M T W 9 / P L growth activity was observed just before the salt front. Clearly, an inhibitor to M T W 9 / P L cell growth was present and was separated from the mitogenic activity of P- 100 chromatography. Additionally, we have examined the low molecular weight fractions containing M T W 9 / P L activity for a possible inhibitor to 3T3 cell growth. These experiments (data not shown) revealed no 3T3 cell inhibitor in the low molecular weight range, indicating a clear separation of low molecular weight M T W 9 / P L activity from PDGF. DISCUSSION One of the major findings of the work reported here is that lysates of h u m a n platelets contain mitogenic activity for a considerable n u m b e r of different types of cells that were originally derived from several species. Indeed, cells from human,
rat, mouse, Syrian hamster, Chinese hamster, and monkey all showed growth responses to h u m a n platelet lysates. These data have led us (19) to suggest that platelet mitogens may have a broad physiological significance. While these observations themselves do not prove that the platelet mitogens are involved in physiological functions in tissues such as brain, liver, kidney, m a m mary, pituitary, adrenal cortex, or connective tissue, the finding of such a broad range of growth responses with all of these suggests possible new implications. If the mitogenic activity of platelets is limited to only a few molecular species, it seems that the response to these mitogens is conserved in many species and differentiated cell types. Since platelet aggregation or release, or both, is not presently thought to be involved in the normal adult function of m a n y of these tissues, could it be that the role of these mitogens was originally in embryogenesis or at some stage of development prior to reaching the adult? This might mean that adult tissues have a residual responsiveness to these factors. Another possibility is that these factors are not always associated with the platelet. At present, the only proposed function of platelet mitogens
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703
PLATELET-DERIVED GROWTH FACTORS
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FIG. 4. Elution profile of Biogel P-100 chromatography of crude platelet-derived growth factor (6 mL 6.7 mg/mlh prepared as described in Materials and Methods, was applied to a 1.5 x 88 cm of Biogel ])-100 equilibrated with PBS. Fractions (4.0 ml) were collected and assayed at 280 nm for protein concentration and 0.3 ml of each fraction was added to 2.7 ml of DME and assayed for MTW0/PL cell and BALB/e 3T3 cell growth, as described in Materials and Methods. The elution volume of the following marker proteins are shown above the elution profile: bovine serum albumin tool wt. 67,000), ovalbumin (tool wt, 43,000), and ribonuclease A (mol wt, 13,700).
are in wound healing (i.e., fibroblast growth promotion) and in atherosclerosis (4). Both of these conditions represent tissue damage states. The question raised by our findings is whether there is a normal physiological role of platelet growth factor in vivo that may be either active or passive maintenance of cell functions. The future studies should center on establishing conditions for platelet mitogen release and, most importantly, determining what is the origin of these mitogenic activities. It may be possible that the P D G F activity is stored in platelets but could be produced locally in other tissues where it has a major regulatory role. If the various growth factors are associated with platelets in states other than in the released granules, they may be available under other conditions and have physiological functions unrelated to conditions of platelet aggregation and blood clotting. These questions remain unanswered but are interesting topics for future studies. The last major point made by the experiments presented is that it is possible to separate clearly the 3T3 mouse fibroblast P D G F activity (13) from the activity effective with M T W 9 / P L rat mammary tumor cells. The first separation of two
activities from platelet lysates was shown in the trypsin digestion experiment, where 3T3 P D G F was destroyed whereas the M T W 9 / P L activity was unaffected. The second separation is shown after conducting the initial step (i.e., heating) of the platelet lysate to yield PDGF. The crude P D G F showed no mitogenic activity with M T W 9 / P L cells, but when this material was chromatographed on Biogel P-100, a high and low molecular weight form of M T W 9 / P L cell mitogen was separated from the high molecular weight P D G F for 3T3 cells. Whether the M T W 9 / P L activities at high and low molecular weight are different molecular species or complex aggregation products remains to be established. Nevertheless, we now believe that the demonstration of a separable form of platelet rnitogen for an epithelial origin tumor cell (17) suggests that the hypothesis stated in the Introduction, namely, that mammary tumor growth is potentially promoted by platelet aggregation conditions, gains further support. Some final comments are necessary regarding the potential role of platelet-derived growth factors in mammary tumor growth in vivo. Our experiments have not shown a significant difference
704
EASTMENT AND SIRBASKU
in the rate of growth of epithelial tumor cells in platelet-poor plasma versus serum in vitro. Significant differences are only seen with such untransformed, contact inhibited cells as h u m a n fibroblasts, smooth muscle cells, and 3T3 fibroblasts. Although these data could be interpreted to m e a n that platelet factor is not required for epithelial t u m o r growth in vivo, there are some important other interpretations to consider. It is well known that m a n y cell lines respond to more than one type of mitogen. The best example is 3T3 cells that respond to fibroblast growth factor, hydrocortisone, prostaglandins, epidermal growth factor, and P D G F . While the fibroblast in vivo may not see all these agents at one time, one or another could be selectively elevated or taken up from plasma under different physiological conditions, or both. Similar circumstances could be important for growth of such tumors as the estrogen-responsive M T W 9 / P L in vivo. Factors from both plasma and platelets could be important depending upon local concentration, the permeability of the capillary network, and the estrogen level in the blood. It would seem that the resolution of these questions with regard to estrogen-responsive m a m m a r y tumor cell growth in vivo will come when sufficient purified material is available to conduct growth factor localization studies in vivo and when antibody raised to the activity can be used to attempt to influence t u m o r growth in vivo. F r o m the present data, the most certain conclusion we can arrive at is that the distinction between platelet-derived competence factors and plasma-derived progression factors will be difficult to sort out with tumor cells. They do not reach a quiescent state, but instead, exist in a condition of continuous cell cycle traverse. Indeed, the separable phenomena of competence and progression may be lost in tumor cells.
REFERENCES 1. Ross, R.; Biomset, J.; Kariya, B.; Harker, L. A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc. Natl. Acad. Sci. U.S.A. 71: 1207-1210; 1974. 2. Kohler, N.; Lipton, A. Platelets as a source of fibroblast growth promoting activity. Exp. Cell. Res. 87: 297-301; 1974. 3. Balk, S. D. Calcium as a regulator of the proliferation of normal, but not of transformed, chicken fibroblasts in a plasma-containing medium. Proc. Natl. Acad. Sci. U.S.A. 68: 271-275; 1971.
4. Westermark, B.; Wasteson, A. A platelet factor stimulating human normal glial cells. Exp. Cell. Res. 98: 1970-1974; 1976. 5. Heldin, C.; Wasteson, A.; Westermark, B. Partial purification and characterization of platelet factors stimulating the multiplication of normal human glial ceils. Exp. Cell Res. 109: 429-437; 1977. 6. Kcpner, N.; Creasy, G.; Lipton, A. Platelets as a source of cell-proliferating activity. Platelets: A multi-disciplinary approach, de Gaetano, G.; Garattini, S. eds. New York: Raven Press; 1978: 205-212. 7. Antonaides, H.; Scher, C. Radioimmunoassay of a human serum growth factor for BALB/c 3T3 cells: Derivation from platelets. Proc. Natl. Acad. Sci. U.S.A. 74: 1973-1977; 1977. 8. Pledger, W.; Stiles, C.; Antoniades, H.; Scher, C. Induction of DNA synthesis in BALB/c 3T3 cells by serum components: Reevaluation of the commitment process. Proc. Natl. Acad. Sci. U.S.A. 74: 4481-4485; 1977. 9. Pledger, W.; Stiles, C.; Antoniades, H.; Scher, C. An ordered sequence of events is required before BALB/c 3T3 cells become committed to DNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 75: 2839-2843; 1978. 10. Vogel, A.; Raines, E.; Kariya, B.; Rivet, M.; Ross, R. Coordinate control of 3T3 cell proliferation by platelet-derived growth factor and plasma components. Proc. Natl. Acad. Sci. U.S.A. 75: 2810-2814; 1978. II. Stiles, C.; Capone, G.; Scher, C.; Antoniades, H.; VanWyk, J.; Pledger, J. Dual control of (:ell growth by sonlatomedins and platelet-dcrived growth factor. Proc. Natl. Acad. Sci. U.S.A. 76: 1279-1283; 1979. 12. Witte, L. D.; Kaplan, K.; Nossel, H.; Lagcs, B.; Weiss, H.; Goodman, D. Studies of the release from human platelets of the growth factor for cultured human arterial smooth muscle cells. Circ. Res. 42: 402-409; 1978. 13. Antonaides, H. N.; Scher, C.; Stiles, C. Purification of human platelet-derived growth factor. Proc. Natl. Acad. Sci. U.S.A. 76: 1809-1813; 1979. 14. Eastment, C.; Sirhasku, D. Platelet lysate preparations contain mitogenic activity for an established rat mammary tumor cell line (abstr.). In Vitro 13: 106; 1977. 15. Eastment, C.; Sorrentino, J.; Sirbasku, D. Expired human platelets as a source of growth factor(s) for cultured cells from several species and tissues (abstr.). In Vitro 14: 343; 1978. 16. Eastment, C.; Sirbasku, D. Platelet derived mammary tumor growth factor. J. Cell. Physiol. 97: 17-27; 1978. 17. Sirbasku, D. Hormone-responsive growth in vivo of a tissue culture cell line established from the MTW9A rat mammary tumor, Cancer Res. 38: 1154-1165; 1978. 18. Gardikos, C.; Arapakis, G.; Dervenoges, S. The effect of certain hormones in platelet aggregation in vitro. Acta Haemat. (Basel) 47: 297-302; 1972.
PLATELET-DERIVED GROWTH FACTORS 19. Clayman, S.; Gadd, R. E. A.; Herbert, D. The in vitro effects of several progestogens, estrogens and non-steroidol compounds with estrogenic activity on adenosine diphosphate induced guineapig platelet aggregation. Experientia 29: 95-96; 1973. 20. Eisen, M.; Napp, A. E.; Vock, R. Inhibition of platelet aggregation caused by estrogen treatment in patients with carcinoma of the prostrate. J. Urol. 114: 93-97; 1975. 21. Mitchell, H . C . Effects of estrogens and a progestogen on platelet adhesiveness and aggregation ha rabbits. J. Lab. Clin. Med. 83: 79-89; 1974. 22. Gasic, G.; Boettiger, D.; Catalfamo, J.; Gasic, T.; Stewart, G. Aggregation of platelets and cell membrane vesiculation by rat ceils transformed in vitro by Rous sarcoma virus. Cancer Res. 38: 2950-2955; 1978. 23. Sirbasku, D.; Kirkland, N. Control of cell growth. IV. Growth properties of a new cell line established from an estrogen-dependent kidney of the Syrian hamster. Endocrinology 98: 1260-1272; 1976. 24. Sorrentino, J.; Kirkland, W.; Sirbasku, 1). Control of cell growth. I. Estrogen-dependent growth in vivo of a rat pituitary tumor cell line. J. Natl. Cancer Inst. 56:1149-1153; 1q76. 25. Kao, F.; Puck, T. Genetics of somatic mammalian cells. VII. Induction and isolation of nutritional mutants in Chinese hamster cells. Proe,. Natl. Acad. Sci. U.S.A. 60: 1275-1281; Iq68. 26. (;ey, G.; ('offman, W.; Kubicek, M. Tissue culture studies of the proliferatiw~ capacities of cervical carcinoma and normal elfithelinm. Cancer l{es. 12: 264-265; 1952. 27. Ilopps, I1.; Bernheim, B.; Nisalak, A.; Thio, J.; Smadel, J. Biologie characteristics of a eon|innous kidney cell line derived from the African Green Monkey. J. Immuuol. 91 : 416-424; I q63. 28. Jenseu, F.; Girardi, A.; Gilden, R.; Koprowski, tt. Infection of hmnan and simian tissue cuhures with Rous sarcoma virus. Proe. Natl. Aead. Sei. U.S.A. 52-59; 1964. 2q. Klebe, R.; Ruddle, F. Neuroblastoma: ,Jell cuhure analysis of a differentiating stem cell system (abstr.). J. Cell. Biol. 43: 69a; 196').
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30. Klebe, R.; Chen, T.; Ruddle, F. Controlled production of proliferating somatic cell hybrids. J. Cell. Biol. 45: 74-82; 1970. 31. Black, P.; Rowe, W. SV-40 induced proliferation of tissue culture cells of rabbit, mouse, and porcine origin. Proc. Soc. Exp. Biol. Med. 114: 721-727: 1963. 32. Brenda, P.; Lightbody, J.; Sato, G.; Levine, L.; Sweet, W. Differentiated rat glial cell strain in tissue culture. Science 161: 370-371; 1968. 33. Buonassisi, V.; Sato, G.; Cohen, A. Hormoneproducing cultures of adrenal and pituitary tumor origin. Proc. Natl. Acad. Sci. U.S.A. 48: 1184-1190; 1962. 34. Yasumura, Y.; Tashjian, A. H.; Sato, G. Establishment of four functional, clonal strains of animal cells in culture. Science 154:1186-1189; 1966. 35. Shin, S.; Yasumura, Y.; Sato, G. Studies on interstitial cells in tissue culture. II: Steroid biosynthesis by a clonal line of rat testicular interstitial cells, Endocrinology 82: 614-630; 1968. 36. nacpherson, I.; Stoker, M. Polyoma transformation of hamster cell clones - - an investigation of genetic factors affecting cell competence. Virology 16:147-151 ; 1962. 37. Aaronson, S.; Todaro, G. Development of 3T3-1ike lines from BALB/c mouse embryo cultures: Transformation susceptibility to SV40. J. Cell. Physiol. 72: 141-148; 1969. 38. tisu, T.; Zenzes, M. Mammalian chromosomes in vitro. SVII ldiogram of the Chinese hamster. J. Natl. Cancer inst. 32: 857-869; 1964. 3q. Coon, 11. Chmal culture of differentiated rat liver (Jells qabstr. ). 32: 20a; 1()68. 40. Soul(;, If.; Vazquez, J.; Long, A.; Ahert, S.: Breuuau, M. A human cell line from a l)leltral effusion derived from a breast carcinoma. J. Natl. Cancer Inst. 51 : 1409 1413; 1973. 41. Stiles, C.; Pledger, W.; VanWyk, J.; Autoniades, 11.; Scher, C. Serum components whieh regulate the commitment of BAI,B/c 3T3 ('.ells to I)NA synthesis, lhwnmnes and Cell Culture - - A Tribute to Gordon Tomkins. Sato, G.; l{oss, R. eds. New York: Cold Spring Itarbor Laboratory, Cold Spring IIarhor; 1979. 42. Gospodarowicz, 1). Purification of a fibroblast growth factor from bovine pituitary. J. Biol. Chem. 250:2515-2520; 1975.
T h e authors would like to a c k n o w l e d g e the expert technical assistance of M r s . F r a n c e s Leland. T h i s work was s u p p o r t e d by American C a n c e r Society G r a n t BC-255B a n d N I H G r a n t C A 26617.
