Int. .I Cancer: . 46, 858-863 (1990) 0 1990 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I'Union Internationale Contre 1e Cancer
DETECTION OF COLONIC GROWTH FACTORS USING A HUMAN COLONIC CARCINOMA CELL LINE (LIM 1215) R.H. WHITEHEAD', E.C. NICE,C.J. LLOYD,R. JAMESand A.W. BURGESS Ludwig Institute for Cancer Research, Melbourne Tumor Biology Branch, Victoria 3050, Australia. Although the colonic mucosa is one of the most rapidly proliferating epithelial tissues in the body, little is known about the factors that direct this proliferation. In this report we have studied the parameters of both a mitogenic and a clonogenic assay for detecting potential colonic growth factors (CGF). Using a colon carcinoma cell line (LIM I215), which has retained a number of the properties of normal colonic mucosa, we have assayed a range of mitogenic factors for CGF activity. lH-thymidine incorporation by the LIM I21 5 cell line was stimulated by low concentrations of epidermal growth factor and basic fibroblast growth factor and, to a lesser extent, by higher concentrations of interleukin-I and insulin-like growth factor I. The cells did not respond to a range of other mitogens and lymphokines. Optimal clonogenic response in a soft-agar assay was obtained using a primary pituitary extract.
Although the colonic mucosa is one of the most rapidly proliferating epithelial tissues in the body (Potten et a l . , 1982; Wright et al., 1975) very little is known about the factors that control the proliferation and differentiation of the stem cells in the crypts of Lieberkuhn. A number of growth factors and pharmacological agents have been reported to influence the proliferation of the colonic mucosa. Epidermal growth factor (EGF) (Al-Nafussi and Wright, 1982; Malo and Menard, 1982; Ulshen et nl., 1986), gastrin (Johnson and Guthrie, 1976; Johnson, 1982) and biogenic amines (Tutton and Barkla, 1987) have all been reported to induce colonic mucosal proliferation in viva. In vitro studies have utilized colon carcinoma cell lines from humans or rodents and have shown that these cell lines respond to some peptide hormones e.g., EGF, transforming growth factor a (TGFa) and gastrin (Brattain et al., 1981, 1984; Maley et al., 1986; Conteas and Nandi Majumdar, 1986). Knowledge of these factors and their mechanism of action may aid in our understanding of the process that leads to the appearance of hyperplastic cells in polyps and of malignant cells in colonic carcinoma. We have previously described a human colon carcinoma cell line (LIMl215; Whitehead et al., 1985) that has retained the ability to differentiate in vitro as indicated by the formation of a typical brush border and which has retained some of the properties of normal mucosa in that the cells produce alkaline phosphatase, dipeptidases and mucin (Whitehead et al., 1985). The growth and differentiation of this cell line is also modulated by the addition of short-chain fatty acids such as butyric acid (Whitehead et al., 1986). In this report, we describe the responsiveness of this cell line to known mitogens and its use as the basis of an assay to detect potential mitogens in tissue extracts derived from porcine colonic mucosa. This material is being studied in the hope that the mucosa might contain endogenous growth factors.
p,g/ml hydrocortisone and 5% fetal calf serum (FCS). The cells were passaged, when confluent, using 0.1% trypsin-0.02% EDTA solution, at a split ratio of 1:3. The cell line was used for assays between passages 15 and 25 with fresh stocks being thawed from liquid N, when the cells reached passage 25, because it was found that the background counts (3H-thymidine, 3H-TdR, incorporation into control wells) tended to increase with prolonged passage. Mitogenic assay The LIM1215 cells were cloned in semi-solid agar using the agar cloning method described below. A number of individual colonies were picked, transferred to small culture vessels and grown to confluence. These clones and the parent line were examined for responsiveness to epidermal growth factor (EGF) and the most responsive clone, clone 1A1, was used in all subsequent mitogenic assays. For assay, the cells were trypsinized, washed once in RPMI 1640, resuspended at a concentration of 2.5 X lo5 cells per ml in RPMI 1640 plus 0.5% FCS and plated in 0.2-ml volumes in 96-well plates. The cultures were incubated overnight at 37°C to allow the cells to attach and the factors to be tested were then added to the wells. Serial dilutions were tested in triplicate for each factor, with all factors being tested at 5 or more dilutions. The plates were then incubated for 22 hr at 37°C in an atmosphere of 5% CO,. After this period, 10 p.1 of 3H-TdR (6.7 Curies/mM, Amersham Australia, Sydney) were added to each well to give a final concentration of 0.5 p,Ci of 3H-TdR per well and the plates were re-incubated for 2 hr. The cells in each well were harvested by removing the medium and washing in phosphate-buffered saline (PBS) , followed by solubilization with a solution of 0.1% (w/v) sarcosyl (Sigma, St Louis, MO) in PBS-EDTA buffer (PH 8.0). The cells were then collected onto glass-fiber filter mats and dried, then scintillation fluid was added and the incorporated radioactivity determined using a liquid scintillation counter. Results were expressed as a stimulation index (SI) calculated as a ratio of the mean of the counts in test wells to the mean of the counts in control wells which contained only cells and medium. The effectiveness of the different growth factors tested was measured by the concentration of factor yielding 50% of maximal stimulation (S15,,). Clonogenic assay The parent cell line (LIM1215) was used for all cloning assays. The cells were cloned at a final concentration of 5,000 cells per ml using a double-layer agar technique with an underlay of 1.5 ml of 0.5% agar in RPMI 1640 and an overlay of the cells in 2 ml of 0.3% agar in RPMI 1640 plus 10% FCS. Potential growth factors were mixed into the cell-medium-agar overlay, at appropriate concentrations, before plating. After the agar had set, the plates were placed in plastic containers and gassed with an atmosphere of 5% CO, in air before incu-
MATERIAL AM) METHODS
Cell line The LIM1215 cell line has been described in detail (Whitehead et al., 1985). The cells, which have a unique karyotype (46, XY, - 13, + der(13)t(1;13) (q32.l;pll) (Jenkyn et al., 1987), were cultured in RPMI 1640 plus 1 p,g/ml insulin, 0.6
'To whom correspondence and reprint requests should be sent, at the Ludwig Institute for Cancer Research, P.O. Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia. Received: April 17, 1990 and in revised form July 2, 1990.
859
MITOGENIC STIMULATION OF COLONIC CELL LINE
bating at 37°C for 14 days. To a.id in counting, the colonies were stained with 0.1% crystal violet. The plates were examined using a dissecting microscope and colonies were scored if they contained more than 40 cellls. A crude pituitary extract was included in all assays as a positive control, as previous studies had shown that this pituitary extract induced more colonies than any other factor tested. Growthfactors Murine epidermal growth factor (EGF) was purified from murine salivary glands using a combination of reverse-phase and anion exchange HPLC (Burgess et al., 1982, 1983). Ovine basic fibroblast growth factor (bFGF) was prepared from pituitary glands (Simpson et al., 198'1). A crude extract of bovine pituitaries (A2), containing FGF and other mitogens was prepared by ammonium sulphate precipitation as described by Smith et al. (1984). A colonic mitogen extract (CME) was prepared from porcine colonic mucosa (200 g) by homogenization in 5 vol of ice-cold ammonium carbonate buffer (50 mM, PH 9.2) containing the proteolytic inhibitors pepstatin (3 mg/l) and PMSF (22 mg/l), followed by centrifugation at 10,000 g for 1 hr to remove cell debris. This material could be concentrated by ammonium sulphate pirecipitation by adjusting the mixture to 80% saturation with solid ammonium sulphate. Following centrifugation, the resultant pellet was redissolved in 20 ml of Milli-Q water and 2.5-ml aliquots were desalted using gel filtration on a PD-10 columin (recovery volume 3.5 ml) prior to assay. Nerve growth factor (NGF) was a gift from Dr. P. Bartlett, Walter and Eliza Hall Institute, Melbourne, whilst plateletderived growth factor (PDGF) was obtained from Dr. C. Heldin, Ludwig Institute for Cancer Research, Uppsala, Sweden. Insulin-like growth factors (IGF-I and 2) were provided by Dr. H. Jonas, University of Melbourne. Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was from Schering-Plough, Union, NJ (Dr. T. Nagabhusan). Recombinant human granulocyte colony-stimulating factor (GCSF) was from Amgen, Thousand Oaks, CA (Dr. L. Souza). Macrophage colony-stimulating factor (M-CSF) was prepared as described by Burgess et al. (1985). Recombinant interleukins (IL-1,2,3,4,5 and 6) were provided by Dr. B. Pike, Walter and Eliza Hall Institute, Melbourne. Leukemia inhibitory factor (LIF) was supplied by Dr. N. Cough, Walter and Eliza Hall Institute, Melbourne. All other peptides were purchased from Sigma. Quantitation EGF and bFGF prepared in our laboratories were quantitated by amino-acid analysis. Samples were hydrolysed in vucuo with gaseous HCl generated from 6~ HCl containing 0.1% (w/v) phenol. Norleucine was added as an internal standard to enable accurate quantitation to be made. Analyses were performed on a Beckman Model 6300 analyser equipped with a SECA integrator. The pituitary extract (A2) and the porcine colonic mucosal extract (CME) were quantified by the Lowry method.
RESULTS
Mitogenic assay Selection of the most responsive LIM1215 clone. A total of 12 clones were assayed in the mitogenic assay for responsiveness to EGF, FGF and a pituitary extract (A2). Of the clones showing a good responsiveness to all 3 mitogens, clone 1Al was chosen for use in the mitogenic assay (Table I) because of its high level of responsiveness to the pituitary extract. Some intra-assay variation in the stimulation index was observed; however the SI was usually between 2 and 4. The variation between replicate wells for the same mitogen concentration was normally less than 10%. Optimization of assay conditions Preliminary studies using EGF as the mitogen demonstrated that 0.5% FCS was the minimal concentration of FCS for maximal stimulation of 3H-TdR uptake (Fig. la). When the optimal time of incubation was investigated it was found that addition of 3H-TdRfor a period of 2 hr after 22 hr of incubation gave optimal incorporation. Labelling for longer periods did not increase the stimulation index (Fig. lb). Similar studies on the optimal cell concentration per well indicated that 5 X 104 cells per well gave the optimal responses to the mitogens being studied. At cell concentrations below this level, incorporation was low and results were variable. At cell concentrations above this value, autocrine effects were noted which tended to mask specific responses to the mitogens as the cells incorporated high levels of 3H-TdR even in the control cultures. Responsiveness to potential colonic mitogens Using the assay conditions described above, LIM1215 cells responded well to both EGF and FGF, with the concentration of factor giving the SI,, being 100 pg/ml for EGF and 300 pg/ml for bFGF (Fig. 2). The cells also responded to mitogens in the pituitary (A2) and colonic mucosal extracts (Fig. 3). While both of these extracts were inhibitory at high protein concentrations, the pituitary extract stimulated half maximal response at 2 pg/ml and the colonic mucosal extract at 20 pg/ml . The cells were also tested against a range of other known mitogens. Of these mitogens, only interleukin- 1 and insulinlike growth factor-1 induced stimulation in the LIM1215 cells. The maximal stimulation obtained with IL-1 was only 50% of that achieved with EGF or bFGF with an SI,, concentration of 2 ng/ml. The SI,, concentration of IGF-1 was 50 ng/ml, which is 500-fold higher than the SI,, concentration for EGF (Table 11). The maximal stimulation obtained with IGF-1 was only 20% of the maximal stimulation obtained with EGF. Eight gastrointestinal peptides (bombesin, cholecystekinin, gastrin, glucagon, peptide YY, secretin, somatostatin and substance P) were also tested in the mitogenic assay. Each peptide ; was tested over a concentration range of 10 PM to 1 p ~however, the LIM1215 cells showed no responsiveness to any of these factors in this assay. Clonogenic assays Initial studies of colony formation using different cell con-
TABLE: 1 - STIMULATION OF LIM1215 PARENT LINE AND CLONES BY GROWTH FACTORS Factor
Parent
EGF' FGF
2.22 1.6 3.3
A2
line
Clones IAl
I D6
2A3
2B6
2C5
ACA
ACB
2D6
2C3
LA3
2.9 2.2 4.0
2.3 1.5 2.1
2.9 2.1 3.2
1.9 1.4 2.4
1.7 1.4 1.9
2.1 1.7 2.3
2.5 2.0 3.2
2.8 2.0 2.1
2.1 1.5 2.2
2.4 1.8 1.9
'EGF and bFGF were used at a concentrairion of 1 ng/ml. A2 was used at a concentration of 50 pg/ml.-'Stimulation index of 'H-TdR uptake compared to control wells.
860
WHITEHEAD ET AL.
A
B
0.25
0.5
0.6
0.8
16-16
1.0
18-20 20-22 22-24 24-26
Labelling Period (hours) Serum Concentration (%v/v) FIGURE 1 - ( a ) 3H-TdR incorporation by LIM1215 cells in RPMI 1640 medium containing various concentrations of fetal calf serum. After incubation for 22 hr, the cells were labelled with 3H-TdRfor 2 hr. Results are expressed as stimulation index as described in the “Material and Methods”. (b) 3H-TdR incorporation of LIM1215 cells after a 2-hr labelling period following incubation at 37°C for varying periods of time.
f Colon
1
10
100
1,000
10,000
0.1
1
10
1,000
100
Mitogen Concentration (pghl) FIGURE 2 - Response of LIM1215 cells to a range of concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) in the mitogenic assay.
added (pghl) FIGURE 3 - Response of LIM1215 cells to a range of concentrations of either a pituitary extract (A2) or a pig colonic mucosal extract in the mitogenic assay.
centrations showed that the LIM1215 cell line did not form any colonies if plated at less than 2 X lo3 cells per ml. At cell concentrations above 5 X 104 cells per ml, too many colonies for accurate counting were formed. At a concentration of 5 X lo3 cells per ml (1 X lo4 per plate) the LIM1215 cells cloned in semi-solid agar with a cloning efficiency of 0.6%. The cells responded to the addition of EGF, bFGF and A2 by a significant increase in the cloning efficiency. The pituitary extract (A2) caused a much larger increase in the number of colonies than either EGF or bFGF (Fig. 4). All of the growth factors listed in Table I1 were also tested in this assay; however, none of them induced any increase in the cloning efficiency of these cells. Similarly, none of the gastrointestinal peptides tested in the mitogenic assay were active in the clonogenic assay. Extracts of pig colonic mucosa also stimulated clonogenicity (Fig. 4)of LIM1215 cells except at high concentrations where the extract was inhibitory. Following ammonium sulphate precipitation, this inhibitory effect was no longer apparent. Further fractionation of this material has revealed a number of potential colonic mitogens.
berkuhn has been well documented (Potten et al., 1982; Chang and Leblond, 1971; Cheng and Leblond, 1974). In the parallel situation in the bone marrow, where all the mature cells of the
DISCUSSION
The derivation of the cells of the gastrointestinal mucosa from stem cells resident near the base of the crypts of Lie-
Amount
TABLE I1 - EFFECT OF GROWTH FACTORS/CYTOKINESON 3H-TdR INCORPORATION B Y LIM 1215 CELLS
Growth factors
Interleukin-2 Interleukin-2 Interleukin-3 Interleukin-4 Interleukin-5 Interleukin-6 GM-CSF G-CSF M-CSF EGF bFGF PDGF NGF Insulin-like growth factor-1 (IGF- 1) Insulin-like growth factor-2 LIF
Response
+ (2)’3*
NR (300)3 NR (30) NR (13) NR (6) NR (6) NR (300) NR (60) NR (300) (O.lj4 (0.3)4 NR (300) NR (15)
+ +
+ (50)5
NR (1.5) NR 1100)
’Concentration of mitogen (nglml) which stimulated 50% maximal thymidine incorporation.-2Maximum stimulation by IL-1 was only 50% of the stimulation achieved with EGF.-3NR, not responsive (highest concentration tested in the assay). -4See detailed results in Figure 2.-5Maximum stimulation by IGF-I was only 20%of the stimulation achieved with EGF.
861
MITOGENIC STIMULATION OF COLONIC CELL LINE
A
z
-c 11 U
C 0 ._
I
m -
z
._
tj 6
1
30
100
300
1000
3003
10030
100
1OW
300
3000
10000
FGF Concentration (pglml)
EGF Concentration (pgml)
D
C I -g - 11 0 C
.I
m -
i
._
n ti
1 3
10
30
100
300
A2 Concentration ( pglml)
0.2
0.6
2.0
6.0
20
60
Protein added (pg/ml)
FIGURE4 - Response of LIM1215 to a range of concentrations of mitogens in the clonogenic assay. ( a ) EGF; (b)FGF; ( c ) pituitary extract (A2); (d) pig colonic mucosal extract and an extract of the pellet recovered after precipitation with 80% ammonium sulphate.
hemopoietic system are derived from stem cells resident in the bone marrow, specific factors have been described that control the proliferation and differentiation of the cells of the different hemopoietic lineages. Nothing is known as yet about the factors that may act in this manner to stimulate cell proliferation in the colon. Various factors have been described that can alter the proliferation of the colonic mucosa in vivo; however, the effects seen appear to depend on the age of the animal, the route of administration of the factor and the nutritional status of the animal (Malo and Menard, 1982;Dembinski et al., 1982;Oka et al., 1983;Goodlad et al., 1987).In in vivo studies it is also very difficult to determine whether the effects seen are a primary effect of the factor or whether the factors administered are acting indirectly by inducing a colon-specific mitogen. Induction of a second mitogen has been well demonstrated by Sagor et al. (1985),who administered somatostatin or bombesin to rats in order to modulate the plasma levels of enteroglucagon (another proposed intestinal mitogen). In vitro studies have demonstrated that EGF is a mitogen in organ culture studies of fetal human and mouse intestine (Beaulieu et al., 1985; Finney et al., 1987). In addition, a number of human colonic carcinoma cell lines have been shown to be responsive to EGF in vitro. Not all carcinoma cell lines are responsive to EGF even though a number of lines have been shown to secrete TGFdEGF-like activity in culture (Coffey et al., 1986;Anzano et al., 1989).Brattain et al. (1981,1984)classified the colon carcinoma cell lines that they studied by their dependence on EGF for growth. Coffey et al. (1986)showed that the colon carcinoma cell lines in their study did not respond to EGF in a colony assay. In this study we have defined the responsiveness of one colon carcinoma cell line to a range of growth factors and have
used this cell line to demonstrate the presence of potential mitogens in the colonic mucosa and pituitary. The LIM1215 cells have proved to be a suitable cell line for assaying for the presence of colonic mitogens. The cells respond to EGF and bFGF in both mitogenic and colony assays. The cells were also responsive to IL-1 and IGF-1 in the mitogenic assay with ECso concentrations of 2 ng/ml and 50 ng/ml, respectively. Although the cell line was responsive to these 2 factors, the maximal stimulation obtained was less than that obtained with either EGF or bFGF. IGF-1 is a known mitogen for a range of cell types. Enhanced levels of messenger RNA for IGF-1have been demonstrated in colon carcinoma (Tricoli et al., 1986)suggesting that IGF-1 might be an autocrine factor for colon carcinoma cells. 1L-1 stimulates both metabolic changes and proliferation in a wide range of cell types (Schindler and Dinarelloca, 1990). Of particular relevance are the reported effects of IL-1on the stimulation of hepatic protein synthesis (Andus et al., 1988) and the proliferation of keratinocytes (Morhenn et al., 1989). IL-1 is an extremely potent inducer of other cytokines and it will be necessary to determine whether it exerts its effects directly or via the induction of a direct mitogen. The range of growth factors capable of stimulating these colonic cells appears to be limited and there is a structural resemblance between both aFGF and IL-1 (Thomas et al., 1985) and their receptors (Sims et al., 1988).Perhaps there is some common factor in the mechanisms by which these regulators stimulate the growth of colonic cells. The mitogenic effects of IL-1on the colonic cells are interesting in view of the infiltration of T-lymphocytes into the intestinal mucosa during inflammatory bowel disease (MacDonald and Spencer, 1988). The possible role of T-lymphocytes and lymphokines in the control of intestinal cell production warrants further investigation.
862
WHITEHEA D ET AL.
The fact that the cells clone more efficiently in response to the pituitary extract than in response to either EGF or bFGF suggests that another mitogen may be present in this extract. It is not known if this factor is a mitogen as improved cloning efficiency does not represent mitogenesis but rather is an indication of increased recruitment to growth. The ability to grow in soft agar is considered to be an indication of a cell’s ability to grow autonomously and the unknown pituitary factor is able to increase the proportion of LIM1215 cells capable of growing in this way. Pituitary extract contains bFGF (Esch et al., 1985); however, stimulation seen with this extract was always greater than that obtained with purified bFGF even at maximal concentrations. Even the use of combinations of EGF and FGF in the clonogenic assay did not lead to an increase in colony numbers to the levels obtained with the pituitary extract. However, to obtain meaningful results, the parameters of these assays must be carefully controlled as autocrine stimulation with subsequent increase in baseline 3H-TdR incorporation and colony numbers occurs once the cell concentration reaches a critical level. This reduces the stimulation index, thus ren-
dering the cells less useful in this type of assay. The finding of autocrine stimulation at high cell concentrations is not surprising as the LIM1215 cells possess the TGFa message (Malden et al., 1989). The finding of mitogenic activity for a colonic carcinoma cell line in a crude extract of pig colonic mucosa indicates that the factors controlling the proliferation of the colonic mucosa may be intrinsic to the tissue. This finding is being explored further. Our report shows that the assays described might be useful in monitoring the purification of the mitogenic factor(s) of the pig colonic mucosa and the clonogenic factor(s) present in the bovine pituitary extract. ACKNOWLEDGEMENTS
We thank Mrs. J. Winikoff, Ms. D. Holt, Ms. L. Angus and Mr. L. Fabri for excellent technical assistance. The assistance of Dr. R. Simpson and Mr. G. Reid with the amino-acid analysis is gratefully acknowledged.
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KLEPPER,GOSPODAROWICZ, D., BOHLEN, R. and GUILLEMN ,R.,Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc. nut. Acad. Sci. (Wash.), 82, 6507-6511 (1985). FINNEY,K.J., INCE,P., APPLETON, P., SUNTER,T.P. and WATSON,A.J., A trophic effect of EGF on rat colonic mucosa in organ culture. Cell Tiss. Kinet., 20, 43-56 (1987). GOODLAD, R.A., WILSON,T.J.G., LENTON,W., GREGORY, H., MCCULLAGH,K.K. and WRIGHT,N.A., Intravenous but not intragastric urogastrone-EGF is trophic to the intestine of parenterally fed rats. Gut, 28, 573-582 (1987). JENKYN, D.J., WHITEHEAD, R.H., HOUSE,A.K. and MALEY,M.A.L., Single chromosome defect, partial trisomy Iq, in a colon cancer cell line. Cancer Genet. Cyrogenet., 27, 357-360 (1987). JOHNSON, L.R., Effect of exogenous gut hormones on gastrointestinal mucosal growth. Scand. J . Gasrroenterol., 74, 89-92 (1982). JOHNSON, L.R. and GUTHRIE,P.D., Stimulation of DNA synthesis by big and little gastrin (G-34 and ‘3-17). Gastroenterology, 71,599-602 (1976). MACDONALD, T.T. and SPENCER,J., Evidence that activated mucosal T cells play a role in the pathogenesis of enteropathy in human small intestine. J . exp. Med., 167, 1341-1349 (19883. MALDEN,L.T., NOVAK,U. and BURGESS,A.W., Expression of transforming growth factor alpha messenger RNA in the normal and neoplastic gastrointestinal tract. Int. J . Cancer, 43, 380-384 (1989). MALEY,M.A.L., AGREZ,M.V. and HOUSE,A.K., Growth factor-induced proliferative responses of human and DMH-induced rat colorectal tumour cell lines. Ausl. J . exp. Biol. med. Sci.. 64, 4 5 5 4 5 1 (1986). MALO,C. and MENARD,D., Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology, 83, 28-35 (1982). MORHENN,V.B., WASTER,G.J., CUA, A.B. and MANSBRIDGE, J.N., Effects of recombinant interleukin- I and interleukin-2 on human keratinocytes. J . invest. Dermatol., 93, 121-126 (1989). OKA. Y., GHISHAN,F.K., GREENE,H.L. and ORTH, D.N., Effect of mouse epidermal growth factor/urogastrone on the functional maturation of rat intestine. Endocrinology, 112, 94C944 (1983). POTTEN,C.S., CHWALINSKI, S., SWINDELL,R. and PALMER,M., The spatial organisation of the hierarchical proliferative cells of the crypts of the small intestine into clusters of synchronised cells. Cell Tiss. Kinet., IS, 351-369 (1982). SAGOR,G.R., GHATEI,M.A., ~’SHAUGHNESSY, D.J., AL-MUKHTAR, M.Y.T., WRIGHT,N.A. and BLOOM,S.R., influence of somatostatin and bombesin on plasma enteroglucagon and cell proliferation after intestinal resection in the rat. Gut, 26, 89-94 (1985). SCHINDLER, R. and DINARELLOCA, C.A., Interleukin-1. In: A Hakemicht (ed.), Growth factors, diyerentiation factors and cytokines, pp. 85-102, Springer, Berlin (1990). SIMPSON, R.J., MORITZ,R.L., LLOYD,C.J., FABRI, L.J., NICE, E.C., RUBIRA,M.R. and BURGESS, A.W., Primary structure of ovine pituitary basic fibroblast growth factor. FEES Lett., 224, 128-132 (1987).
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SIMS,E.J., MARCH,C.J., COSMAN, D., WIDMER,M.B., MACDONALD,TUTTON, D.H., Biogenic amines as regulators of the P.J.M. and BARKLA, H.E., MCMAHON, C.J., GRUBIN, C.E., WIGNALL, J.M., JACKSON, J.L., proliferative activity of normal and neoplastic intestinal epithelial cells CALL,S.M., FRIEND,D., ALPERT,A.R., GILLIS,S., URDAL,D.L. and (review). Anticancer Res., 7 , 1-12 (1987). DOWER,S.K., cDNA expression cloning of the IL-I receptor, a member L.E. and RAASCH, R.H., Effects of intraluULSHEN,M.H., LYN-COOK, of the immunoglobulin superfamily. Science, 241, 585-589 (1988). minal epidermal growth factor on rnucosal proliferation in the small intesSMITH,J.A., WINSLOW, D.P. and RUDI-AND, P.S., Different growth fac- tine of adult rats. Gastroenterology, 91, 1 1 3 4 1140 (1986). tors stimulate cell division of rat mammary epithelial, myoepithelial, and stromal cell lines in culture. J . cell. Physiol., 119, 32Ck326 (1984). F.A., ST JOHN,D.J.B. and MA,J., A colon WHITEHEAD, R.H., MACRAE, THOMAS, K.A., RIOS-CANDELORE, M., GIMENEZ-GALLEGO, G., DI carcinoma cell line (LIM1215) derived from a patient with inherited nonJ . and FITZPATRICK, S., Pure brain- polyposis colorectal cancer. J . naf. Cancer Insf., 74, 759-765 (1985). SALVO,J . , BENNET,C., RODKEY, derived acidic fibroblast growth factor is a potent angiogenic vascular WHITEHEAD, P.S., Effects of short R.H., YOUNG,G.P. and BHATHAL, endothelial cell mitogen with sequence homology to interleukin- 1 . Proc. chain fatty acids on a new human colon carcinoma cell line (LIM1215). naf. Acad. Sci. (Wash.j, 82, 6409-6413 (1985). Gut, 27, 1457-1463 (1986). TRICOLI. J.V., RALL,L..B., KIARAKOUSIS, C.P., HERRERA, L., PETRELLI, N.J., BELL,(3.1. and SHOWS, T.B., Enhanced levels of insulin-likegrowth WRIGHT,N.A., AL-DEWACHI, H.S., APPLETON,D.R. and WATSON, factor messenger RNA in human colon carcinomas and liposarcomas. Can- A.J., Cell population kinetics in the rat jejunal crypt. Cell Tiss. Kinet., 8, cer Res., 46, 6169-61’73 (1986). 361-368 (1975).
Publication of the International Union Against Cancer Publication de I'Union Internationale Contre 1e Cancer
DETECTION OF COLONIC GROWTH FACTORS USING A HUMAN COLONIC CARCINOMA CELL LINE (LIM 1215) R.H. WHITEHEAD', E.C. NICE,C.J. LLOYD,R. JAMESand A.W. BURGESS Ludwig Institute for Cancer Research, Melbourne Tumor Biology Branch, Victoria 3050, Australia. Although the colonic mucosa is one of the most rapidly proliferating epithelial tissues in the body, little is known about the factors that direct this proliferation. In this report we have studied the parameters of both a mitogenic and a clonogenic assay for detecting potential colonic growth factors (CGF). Using a colon carcinoma cell line (LIM I215), which has retained a number of the properties of normal colonic mucosa, we have assayed a range of mitogenic factors for CGF activity. lH-thymidine incorporation by the LIM I21 5 cell line was stimulated by low concentrations of epidermal growth factor and basic fibroblast growth factor and, to a lesser extent, by higher concentrations of interleukin-I and insulin-like growth factor I. The cells did not respond to a range of other mitogens and lymphokines. Optimal clonogenic response in a soft-agar assay was obtained using a primary pituitary extract.
Although the colonic mucosa is one of the most rapidly proliferating epithelial tissues in the body (Potten et a l . , 1982; Wright et al., 1975) very little is known about the factors that control the proliferation and differentiation of the stem cells in the crypts of Lieberkuhn. A number of growth factors and pharmacological agents have been reported to influence the proliferation of the colonic mucosa. Epidermal growth factor (EGF) (Al-Nafussi and Wright, 1982; Malo and Menard, 1982; Ulshen et nl., 1986), gastrin (Johnson and Guthrie, 1976; Johnson, 1982) and biogenic amines (Tutton and Barkla, 1987) have all been reported to induce colonic mucosal proliferation in viva. In vitro studies have utilized colon carcinoma cell lines from humans or rodents and have shown that these cell lines respond to some peptide hormones e.g., EGF, transforming growth factor a (TGFa) and gastrin (Brattain et al., 1981, 1984; Maley et al., 1986; Conteas and Nandi Majumdar, 1986). Knowledge of these factors and their mechanism of action may aid in our understanding of the process that leads to the appearance of hyperplastic cells in polyps and of malignant cells in colonic carcinoma. We have previously described a human colon carcinoma cell line (LIMl215; Whitehead et al., 1985) that has retained the ability to differentiate in vitro as indicated by the formation of a typical brush border and which has retained some of the properties of normal mucosa in that the cells produce alkaline phosphatase, dipeptidases and mucin (Whitehead et al., 1985). The growth and differentiation of this cell line is also modulated by the addition of short-chain fatty acids such as butyric acid (Whitehead et al., 1986). In this report, we describe the responsiveness of this cell line to known mitogens and its use as the basis of an assay to detect potential mitogens in tissue extracts derived from porcine colonic mucosa. This material is being studied in the hope that the mucosa might contain endogenous growth factors.
p,g/ml hydrocortisone and 5% fetal calf serum (FCS). The cells were passaged, when confluent, using 0.1% trypsin-0.02% EDTA solution, at a split ratio of 1:3. The cell line was used for assays between passages 15 and 25 with fresh stocks being thawed from liquid N, when the cells reached passage 25, because it was found that the background counts (3H-thymidine, 3H-TdR, incorporation into control wells) tended to increase with prolonged passage. Mitogenic assay The LIM1215 cells were cloned in semi-solid agar using the agar cloning method described below. A number of individual colonies were picked, transferred to small culture vessels and grown to confluence. These clones and the parent line were examined for responsiveness to epidermal growth factor (EGF) and the most responsive clone, clone 1A1, was used in all subsequent mitogenic assays. For assay, the cells were trypsinized, washed once in RPMI 1640, resuspended at a concentration of 2.5 X lo5 cells per ml in RPMI 1640 plus 0.5% FCS and plated in 0.2-ml volumes in 96-well plates. The cultures were incubated overnight at 37°C to allow the cells to attach and the factors to be tested were then added to the wells. Serial dilutions were tested in triplicate for each factor, with all factors being tested at 5 or more dilutions. The plates were then incubated for 22 hr at 37°C in an atmosphere of 5% CO,. After this period, 10 p.1 of 3H-TdR (6.7 Curies/mM, Amersham Australia, Sydney) were added to each well to give a final concentration of 0.5 p,Ci of 3H-TdR per well and the plates were re-incubated for 2 hr. The cells in each well were harvested by removing the medium and washing in phosphate-buffered saline (PBS) , followed by solubilization with a solution of 0.1% (w/v) sarcosyl (Sigma, St Louis, MO) in PBS-EDTA buffer (PH 8.0). The cells were then collected onto glass-fiber filter mats and dried, then scintillation fluid was added and the incorporated radioactivity determined using a liquid scintillation counter. Results were expressed as a stimulation index (SI) calculated as a ratio of the mean of the counts in test wells to the mean of the counts in control wells which contained only cells and medium. The effectiveness of the different growth factors tested was measured by the concentration of factor yielding 50% of maximal stimulation (S15,,). Clonogenic assay The parent cell line (LIM1215) was used for all cloning assays. The cells were cloned at a final concentration of 5,000 cells per ml using a double-layer agar technique with an underlay of 1.5 ml of 0.5% agar in RPMI 1640 and an overlay of the cells in 2 ml of 0.3% agar in RPMI 1640 plus 10% FCS. Potential growth factors were mixed into the cell-medium-agar overlay, at appropriate concentrations, before plating. After the agar had set, the plates were placed in plastic containers and gassed with an atmosphere of 5% CO, in air before incu-
MATERIAL AM) METHODS
Cell line The LIM1215 cell line has been described in detail (Whitehead et al., 1985). The cells, which have a unique karyotype (46, XY, - 13, + der(13)t(1;13) (q32.l;pll) (Jenkyn et al., 1987), were cultured in RPMI 1640 plus 1 p,g/ml insulin, 0.6
'To whom correspondence and reprint requests should be sent, at the Ludwig Institute for Cancer Research, P.O. Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia. Received: April 17, 1990 and in revised form July 2, 1990.
859
MITOGENIC STIMULATION OF COLONIC CELL LINE
bating at 37°C for 14 days. To a.id in counting, the colonies were stained with 0.1% crystal violet. The plates were examined using a dissecting microscope and colonies were scored if they contained more than 40 cellls. A crude pituitary extract was included in all assays as a positive control, as previous studies had shown that this pituitary extract induced more colonies than any other factor tested. Growthfactors Murine epidermal growth factor (EGF) was purified from murine salivary glands using a combination of reverse-phase and anion exchange HPLC (Burgess et al., 1982, 1983). Ovine basic fibroblast growth factor (bFGF) was prepared from pituitary glands (Simpson et al., 198'1). A crude extract of bovine pituitaries (A2), containing FGF and other mitogens was prepared by ammonium sulphate precipitation as described by Smith et al. (1984). A colonic mitogen extract (CME) was prepared from porcine colonic mucosa (200 g) by homogenization in 5 vol of ice-cold ammonium carbonate buffer (50 mM, PH 9.2) containing the proteolytic inhibitors pepstatin (3 mg/l) and PMSF (22 mg/l), followed by centrifugation at 10,000 g for 1 hr to remove cell debris. This material could be concentrated by ammonium sulphate pirecipitation by adjusting the mixture to 80% saturation with solid ammonium sulphate. Following centrifugation, the resultant pellet was redissolved in 20 ml of Milli-Q water and 2.5-ml aliquots were desalted using gel filtration on a PD-10 columin (recovery volume 3.5 ml) prior to assay. Nerve growth factor (NGF) was a gift from Dr. P. Bartlett, Walter and Eliza Hall Institute, Melbourne, whilst plateletderived growth factor (PDGF) was obtained from Dr. C. Heldin, Ludwig Institute for Cancer Research, Uppsala, Sweden. Insulin-like growth factors (IGF-I and 2) were provided by Dr. H. Jonas, University of Melbourne. Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was from Schering-Plough, Union, NJ (Dr. T. Nagabhusan). Recombinant human granulocyte colony-stimulating factor (GCSF) was from Amgen, Thousand Oaks, CA (Dr. L. Souza). Macrophage colony-stimulating factor (M-CSF) was prepared as described by Burgess et al. (1985). Recombinant interleukins (IL-1,2,3,4,5 and 6) were provided by Dr. B. Pike, Walter and Eliza Hall Institute, Melbourne. Leukemia inhibitory factor (LIF) was supplied by Dr. N. Cough, Walter and Eliza Hall Institute, Melbourne. All other peptides were purchased from Sigma. Quantitation EGF and bFGF prepared in our laboratories were quantitated by amino-acid analysis. Samples were hydrolysed in vucuo with gaseous HCl generated from 6~ HCl containing 0.1% (w/v) phenol. Norleucine was added as an internal standard to enable accurate quantitation to be made. Analyses were performed on a Beckman Model 6300 analyser equipped with a SECA integrator. The pituitary extract (A2) and the porcine colonic mucosal extract (CME) were quantified by the Lowry method.
RESULTS
Mitogenic assay Selection of the most responsive LIM1215 clone. A total of 12 clones were assayed in the mitogenic assay for responsiveness to EGF, FGF and a pituitary extract (A2). Of the clones showing a good responsiveness to all 3 mitogens, clone 1Al was chosen for use in the mitogenic assay (Table I) because of its high level of responsiveness to the pituitary extract. Some intra-assay variation in the stimulation index was observed; however the SI was usually between 2 and 4. The variation between replicate wells for the same mitogen concentration was normally less than 10%. Optimization of assay conditions Preliminary studies using EGF as the mitogen demonstrated that 0.5% FCS was the minimal concentration of FCS for maximal stimulation of 3H-TdR uptake (Fig. la). When the optimal time of incubation was investigated it was found that addition of 3H-TdRfor a period of 2 hr after 22 hr of incubation gave optimal incorporation. Labelling for longer periods did not increase the stimulation index (Fig. lb). Similar studies on the optimal cell concentration per well indicated that 5 X 104 cells per well gave the optimal responses to the mitogens being studied. At cell concentrations below this level, incorporation was low and results were variable. At cell concentrations above this value, autocrine effects were noted which tended to mask specific responses to the mitogens as the cells incorporated high levels of 3H-TdR even in the control cultures. Responsiveness to potential colonic mitogens Using the assay conditions described above, LIM1215 cells responded well to both EGF and FGF, with the concentration of factor giving the SI,, being 100 pg/ml for EGF and 300 pg/ml for bFGF (Fig. 2). The cells also responded to mitogens in the pituitary (A2) and colonic mucosal extracts (Fig. 3). While both of these extracts were inhibitory at high protein concentrations, the pituitary extract stimulated half maximal response at 2 pg/ml and the colonic mucosal extract at 20 pg/ml . The cells were also tested against a range of other known mitogens. Of these mitogens, only interleukin- 1 and insulinlike growth factor-1 induced stimulation in the LIM1215 cells. The maximal stimulation obtained with IL-1 was only 50% of that achieved with EGF or bFGF with an SI,, concentration of 2 ng/ml. The SI,, concentration of IGF-1 was 50 ng/ml, which is 500-fold higher than the SI,, concentration for EGF (Table 11). The maximal stimulation obtained with IGF-1 was only 20% of the maximal stimulation obtained with EGF. Eight gastrointestinal peptides (bombesin, cholecystekinin, gastrin, glucagon, peptide YY, secretin, somatostatin and substance P) were also tested in the mitogenic assay. Each peptide ; was tested over a concentration range of 10 PM to 1 p ~however, the LIM1215 cells showed no responsiveness to any of these factors in this assay. Clonogenic assays Initial studies of colony formation using different cell con-
TABLE: 1 - STIMULATION OF LIM1215 PARENT LINE AND CLONES BY GROWTH FACTORS Factor
Parent
EGF' FGF
2.22 1.6 3.3
A2
line
Clones IAl
I D6
2A3
2B6
2C5
ACA
ACB
2D6
2C3
LA3
2.9 2.2 4.0
2.3 1.5 2.1
2.9 2.1 3.2
1.9 1.4 2.4
1.7 1.4 1.9
2.1 1.7 2.3
2.5 2.0 3.2
2.8 2.0 2.1
2.1 1.5 2.2
2.4 1.8 1.9
'EGF and bFGF were used at a concentrairion of 1 ng/ml. A2 was used at a concentration of 50 pg/ml.-'Stimulation index of 'H-TdR uptake compared to control wells.
860
WHITEHEAD ET AL.
A
B
0.25
0.5
0.6
0.8
16-16
1.0
18-20 20-22 22-24 24-26
Labelling Period (hours) Serum Concentration (%v/v) FIGURE 1 - ( a ) 3H-TdR incorporation by LIM1215 cells in RPMI 1640 medium containing various concentrations of fetal calf serum. After incubation for 22 hr, the cells were labelled with 3H-TdRfor 2 hr. Results are expressed as stimulation index as described in the “Material and Methods”. (b) 3H-TdR incorporation of LIM1215 cells after a 2-hr labelling period following incubation at 37°C for varying periods of time.
f Colon
1
10
100
1,000
10,000
0.1
1
10
1,000
100
Mitogen Concentration (pghl) FIGURE 2 - Response of LIM1215 cells to a range of concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) in the mitogenic assay.
added (pghl) FIGURE 3 - Response of LIM1215 cells to a range of concentrations of either a pituitary extract (A2) or a pig colonic mucosal extract in the mitogenic assay.
centrations showed that the LIM1215 cell line did not form any colonies if plated at less than 2 X lo3 cells per ml. At cell concentrations above 5 X 104 cells per ml, too many colonies for accurate counting were formed. At a concentration of 5 X lo3 cells per ml (1 X lo4 per plate) the LIM1215 cells cloned in semi-solid agar with a cloning efficiency of 0.6%. The cells responded to the addition of EGF, bFGF and A2 by a significant increase in the cloning efficiency. The pituitary extract (A2) caused a much larger increase in the number of colonies than either EGF or bFGF (Fig. 4). All of the growth factors listed in Table I1 were also tested in this assay; however, none of them induced any increase in the cloning efficiency of these cells. Similarly, none of the gastrointestinal peptides tested in the mitogenic assay were active in the clonogenic assay. Extracts of pig colonic mucosa also stimulated clonogenicity (Fig. 4)of LIM1215 cells except at high concentrations where the extract was inhibitory. Following ammonium sulphate precipitation, this inhibitory effect was no longer apparent. Further fractionation of this material has revealed a number of potential colonic mitogens.
berkuhn has been well documented (Potten et al., 1982; Chang and Leblond, 1971; Cheng and Leblond, 1974). In the parallel situation in the bone marrow, where all the mature cells of the
DISCUSSION
The derivation of the cells of the gastrointestinal mucosa from stem cells resident near the base of the crypts of Lie-
Amount
TABLE I1 - EFFECT OF GROWTH FACTORS/CYTOKINESON 3H-TdR INCORPORATION B Y LIM 1215 CELLS
Growth factors
Interleukin-2 Interleukin-2 Interleukin-3 Interleukin-4 Interleukin-5 Interleukin-6 GM-CSF G-CSF M-CSF EGF bFGF PDGF NGF Insulin-like growth factor-1 (IGF- 1) Insulin-like growth factor-2 LIF
Response
+ (2)’3*
NR (300)3 NR (30) NR (13) NR (6) NR (6) NR (300) NR (60) NR (300) (O.lj4 (0.3)4 NR (300) NR (15)
+ +
+ (50)5
NR (1.5) NR 1100)
’Concentration of mitogen (nglml) which stimulated 50% maximal thymidine incorporation.-2Maximum stimulation by IL-1 was only 50% of the stimulation achieved with EGF.-3NR, not responsive (highest concentration tested in the assay). -4See detailed results in Figure 2.-5Maximum stimulation by IGF-I was only 20%of the stimulation achieved with EGF.
861
MITOGENIC STIMULATION OF COLONIC CELL LINE
A
z
-c 11 U
C 0 ._
I
m -
z
._
tj 6
1
30
100
300
1000
3003
10030
100
1OW
300
3000
10000
FGF Concentration (pglml)
EGF Concentration (pgml)
D
C I -g - 11 0 C
.I
m -
i
._
n ti
1 3
10
30
100
300
A2 Concentration ( pglml)
0.2
0.6
2.0
6.0
20
60
Protein added (pg/ml)
FIGURE4 - Response of LIM1215 to a range of concentrations of mitogens in the clonogenic assay. ( a ) EGF; (b)FGF; ( c ) pituitary extract (A2); (d) pig colonic mucosal extract and an extract of the pellet recovered after precipitation with 80% ammonium sulphate.
hemopoietic system are derived from stem cells resident in the bone marrow, specific factors have been described that control the proliferation and differentiation of the cells of the different hemopoietic lineages. Nothing is known as yet about the factors that may act in this manner to stimulate cell proliferation in the colon. Various factors have been described that can alter the proliferation of the colonic mucosa in vivo; however, the effects seen appear to depend on the age of the animal, the route of administration of the factor and the nutritional status of the animal (Malo and Menard, 1982;Dembinski et al., 1982;Oka et al., 1983;Goodlad et al., 1987).In in vivo studies it is also very difficult to determine whether the effects seen are a primary effect of the factor or whether the factors administered are acting indirectly by inducing a colon-specific mitogen. Induction of a second mitogen has been well demonstrated by Sagor et al. (1985),who administered somatostatin or bombesin to rats in order to modulate the plasma levels of enteroglucagon (another proposed intestinal mitogen). In vitro studies have demonstrated that EGF is a mitogen in organ culture studies of fetal human and mouse intestine (Beaulieu et al., 1985; Finney et al., 1987). In addition, a number of human colonic carcinoma cell lines have been shown to be responsive to EGF in vitro. Not all carcinoma cell lines are responsive to EGF even though a number of lines have been shown to secrete TGFdEGF-like activity in culture (Coffey et al., 1986;Anzano et al., 1989).Brattain et al. (1981,1984)classified the colon carcinoma cell lines that they studied by their dependence on EGF for growth. Coffey et al. (1986)showed that the colon carcinoma cell lines in their study did not respond to EGF in a colony assay. In this study we have defined the responsiveness of one colon carcinoma cell line to a range of growth factors and have
used this cell line to demonstrate the presence of potential mitogens in the colonic mucosa and pituitary. The LIM1215 cells have proved to be a suitable cell line for assaying for the presence of colonic mitogens. The cells respond to EGF and bFGF in both mitogenic and colony assays. The cells were also responsive to IL-1 and IGF-1 in the mitogenic assay with ECso concentrations of 2 ng/ml and 50 ng/ml, respectively. Although the cell line was responsive to these 2 factors, the maximal stimulation obtained was less than that obtained with either EGF or bFGF. IGF-1 is a known mitogen for a range of cell types. Enhanced levels of messenger RNA for IGF-1have been demonstrated in colon carcinoma (Tricoli et al., 1986)suggesting that IGF-1 might be an autocrine factor for colon carcinoma cells. 1L-1 stimulates both metabolic changes and proliferation in a wide range of cell types (Schindler and Dinarelloca, 1990). Of particular relevance are the reported effects of IL-1on the stimulation of hepatic protein synthesis (Andus et al., 1988) and the proliferation of keratinocytes (Morhenn et al., 1989). IL-1 is an extremely potent inducer of other cytokines and it will be necessary to determine whether it exerts its effects directly or via the induction of a direct mitogen. The range of growth factors capable of stimulating these colonic cells appears to be limited and there is a structural resemblance between both aFGF and IL-1 (Thomas et al., 1985) and their receptors (Sims et al., 1988).Perhaps there is some common factor in the mechanisms by which these regulators stimulate the growth of colonic cells. The mitogenic effects of IL-1on the colonic cells are interesting in view of the infiltration of T-lymphocytes into the intestinal mucosa during inflammatory bowel disease (MacDonald and Spencer, 1988). The possible role of T-lymphocytes and lymphokines in the control of intestinal cell production warrants further investigation.
862
WHITEHEA D ET AL.
The fact that the cells clone more efficiently in response to the pituitary extract than in response to either EGF or bFGF suggests that another mitogen may be present in this extract. It is not known if this factor is a mitogen as improved cloning efficiency does not represent mitogenesis but rather is an indication of increased recruitment to growth. The ability to grow in soft agar is considered to be an indication of a cell’s ability to grow autonomously and the unknown pituitary factor is able to increase the proportion of LIM1215 cells capable of growing in this way. Pituitary extract contains bFGF (Esch et al., 1985); however, stimulation seen with this extract was always greater than that obtained with purified bFGF even at maximal concentrations. Even the use of combinations of EGF and FGF in the clonogenic assay did not lead to an increase in colony numbers to the levels obtained with the pituitary extract. However, to obtain meaningful results, the parameters of these assays must be carefully controlled as autocrine stimulation with subsequent increase in baseline 3H-TdR incorporation and colony numbers occurs once the cell concentration reaches a critical level. This reduces the stimulation index, thus ren-
dering the cells less useful in this type of assay. The finding of autocrine stimulation at high cell concentrations is not surprising as the LIM1215 cells possess the TGFa message (Malden et al., 1989). The finding of mitogenic activity for a colonic carcinoma cell line in a crude extract of pig colonic mucosa indicates that the factors controlling the proliferation of the colonic mucosa may be intrinsic to the tissue. This finding is being explored further. Our report shows that the assays described might be useful in monitoring the purification of the mitogenic factor(s) of the pig colonic mucosa and the clonogenic factor(s) present in the bovine pituitary extract. ACKNOWLEDGEMENTS
We thank Mrs. J. Winikoff, Ms. D. Holt, Ms. L. Angus and Mr. L. Fabri for excellent technical assistance. The assistance of Dr. R. Simpson and Mr. G. Reid with the amino-acid analysis is gratefully acknowledged.
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