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[CANCER RESEARCH 44, 3613-3619, August 1984] Distinct High-Performance Liquid Chromatography Pattern of Transforming Growth Factor Activity in Urine of Cancer Patients as Compared with That of Normal Individuals Edward S. Kimball,1 William H. Bohn, Karen D. Cockley, Thomas C. Warren, and Stephen A. Sherwin Laboratory ol Molecular Immunoregulation, Facility, Frederick, Maryland 21701 Biological Response Modifiers Program, Division of Cancer Treatment, National Cancer Institute-Frederick ABSTRACT Reverse-phase high-performance liquid Chromatography (HPLC) performed on urine from cancer patients and normal controls revealed the presence of seven chromatographically distinct peaks of transforming growth factor (TGF) activity, as measured by colony formation of normal rat kidney cells in soft agar. Comparison of urines from normal donors and cancer patients showed no differences in EGF (epidermal growth factor)dependent 0-TGF-like activity but did reveal distinct patterns of EGF-related, EGF-independent o-TGF-like activity. All urine sam Cancer Research absence of EGF; and 0-TGFs, which do not bind to EGF recep tors and must act in synergy with either EGF or a-TGFs to promote anchorage-independent growth (1, 3, 20). Like EGF, aTGFs will catalyze phosphorylation of tyrosine moieties in the EGF receptor (17, 28). TGF activity has been identified in tissue culture supernatants of virally and chemically transformed rodent cells (6,15,16) and from human tumor cell lines (13,22,26). In addition, acid extracts of both neoplastic and normal tissues have been shown to contain TGF activity (3,19, 21, 24). Since TGFs are acid stable, low-molecular-weight peptides, urine has been examined for the presence of TGF activity. Sherwin ef a/. (23) have recently reported elevated levels of M, 30,000 to 35,000 TGF activity in the urine of most cancer patients, whereas this molecular weight form was detected only infrequently, and at low levels, in normal controls. Normal controls as well as cancer patients express a M, 6,000 to 8,000 TGF. In this report, we further characterize by using HPLC the EGFcompeting activity and soft-agar growth-promoting activity found in acidified, concentrated human urine extracts. Reverse-phase HPLC revealed the existence of at least 5 species of EGF-related a-TGF-like activities and a smaller number of non-EGF related 0-TGF-like activities when cancer patients and normal controls were examined, whereas gel filtration earlier had revealed only 2 different urinary TGFs (23, 27). In addition, we noted distinct differences between normal donors and cancer patients when compared with regard to the relative levels of the EGF-related TGF activities, one of which was found to correlate with the high M, 30,000 TGF observed previously (23, 27) in the urine of cancer patients. Furthermore, we observed that the M, 30,000 tumor-associated TGF gradually underwent conversion to a M, ples contained at least two chromatographically distinguishable forms of EGF-dependent TGF activity, eluting from HPLC as broad peaks with 30 and 43% acetonitrile. The remaining five TGFs eluted as sharp peaks with 32, 34, 35, 37, and 38% acetonitrile, demonstrated EGF-competing activity, and thus were functionally related to EGF.,Two of the five EGF-related TGFs were consistently elevated only in the urine of cancer patients and eluted with 32% (TGFA) and 37% (TGFD)acetonitrile. Two of the other EGF-related TGFs, eluting with 34% (TGFB) and 35% (TGFC) acetonitrile, were commonly found in both normals and cancer patients. The fifth EGF-related TGF, TGFE, eluting with 38% acetonitrile, was found only in normal donor specimens. TGFA corresponded to the unique M, 30,000 TGF activity previously identified only in the urine of cancer patients. These observations demonstrate that cancer patients produce high levels of EGF-related TGF activities which can be readily distinguished, using reverse-phase HPLC, from EGF-related TGFs produced by normal individuals. Using a solid-phase com petitive radioreceptor binding assay for EGF, we demonstrated that quantitation of EGF-competing activity is as sensitive and effective as the soft-agar colony formation assay for distinguish ing HPLC profiles of urinary TGF from cancer patients versus 6,000 TGF. that from normal individuals. MATERIALS AND METHODS INTRODUCTION TGFs2 are peptides which reversibly promote anchorage-in dependent growth and colony formation in semisolid medium of nontransformed cells (6, 21). Two functional classes of TGFs have been described (18, 20): EGF-related a-TGFs, which com pete with EGF for binding to EGF receptors (6,18,26) and which can promote growth of normal fibroblasts in soft agar in the 1To whom requests for reprints should be addressed, at Connective Tissue Research, Division of Immunology, Ayerest Laboratories Research, Inc., CN 8000, Princeton, NJ 08540. 2The abbreviations used are: TGF, transforming growth factor; EGF, epidermal growth factor; HEGF, human epidermal growth factor from urogastrone; HPLC, high-performance liquid Chromatography; MeCN, acetonitrile; NRK, normal rat kidney. Received October 17,1983; accepted May 1, 1984. Urine Preparation. Morning void urines were collected from patients with a variety of disseminated cancers and stored at 4°.The patients ranged in age from 18 to 63 years, had received no anticancer therapy for a period of at least 3 weeks, and had normal renal function, as judged by serum creatinine and the absence of proteinuria. Urines were also collected from normal laboratory workers ranging in age from 22 to 49 years and also consisted of morning voids. Normal female donors in this group were nonpregnant. Urine specimens were acidified with glacial acetic acid to a final concentration of 1 M, clarified by centrifugation at 20,000 x g for 30 min, and then filtered through a 0.45-,«mfilter (Nalge Corp., Rochester, NY). Between 20 and 50 ml of acidified urine, contain ing 10 mg of protein, were concentrated to less than 1 ml by ultrafiltration on an Amicon XM-50 membrane (Amicon Corp., Danvers, MA) (M, 50,000 cutoff). The concentrate was dialyzed against 1 M acetic acid in the ultrafiltration cell. This procedure generally yielded 200 to 500 fig of protein in the retained portion and contained almost all of the EGF- AUGUST1984 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1984 American Association for Cancer Research. 3613 E. S. Kimball et al. competing activity and soft-agar growth-promoting activity when com pared to dilution curves for the unconcentrated urine and the Amicon filtrate. The retained portion was then applied to a reverse-phase HPLC column. HPLC. A Waters Associates (Milford, MA) CurpBondapak reverse- phase column (3.9 x 300 mm) was used on an Altex Model 324 HPLC (Beckman Instruments, Palo Alto, CA). Gradient elution over a period of 2 hr at a flow rate of 1 ml/min at room temperature, from 0 to 60% acetonitnle (Burdick and Jackson Laboratories, Inc., Hoffman-LaRoche, Inc.) in 0.05% tnfluoroacetic acid (Pierce Chemical Co.), adjusted to pH 2.5 with ammonium hybroxide, was used to develop the chromatogram after an initial isocratic elution for 5 min with 0.05% trifluoroacetic acid. One-mi fractions were collected. Duplicate aliquots (100 ^l) were taken for EGF competition assays, and SOO-plaliquots were used for soft-agar colony assays, as described below. Soft-Agar Growth Assay. Column fractions were tested singly for the presence of factors capable of stimulating nontransformed NRK (clone 49F) fibroWasts, taken at passages 17 to 19, to grow as colonies in soft agar as described previously (5, 6). A single-cell suspension of approxi mately 10* NRK cells was mixed with the lyophiiized sample to be tested and seeded in 2.0 ml of Dulbecco's modified Eagle's medium containing forming activity that is also present in normal control urine were detected. In order to further characterize such urinary TGF activity and permit comparisons to TGFs secreted by human tumor cell lines in vitro (13, 22,26), acid extracts of human urine from normal controls and patients with disseminated neoplastic disease were examined by reverse-phase HPLC. Representative HPLC profiles of acidified urine extracts, from a normal 38-yearold male control and a 50-year-old male patient with renal cell carcinoma, are shown in Chart 1. Four peaks of soft-agar growthpromoting activity (Chart 1, fop) which coeluted with EGF-competing activity (Chart 1, bottom) were seen in the cancer patient, whereas the normal control contained only 3 such peaks. There were noteworthy quantitative and qualitative differences be tween the normal control and the cancer patient. The normal control showed very little, if any, of the TGF activity (TGFA) which 250 H 10% calf serum (Grand Island Biological Co., Grand Island, NY) plus 0.3% agar (Difco Laboratories, Detroit, Ml) into 60-mm Costar Pétri 40 dishes containing a base layer of the same medium plus 0.5% agar. Plates were refed with 2.0 ml of the same medium plus 0.3% agar at 7 days. Colonies which typically contained 8 to 20 cells (23) were scored in 4 random tow-power fields at both 7 and 14 days. Two independent readings were performed for each assay. NRK cells do not grow as colonies in 0.3% agar except in the presence of soft-agar growthpromoting factors, nor do they form colonies in the presence of EGF at concentrations as high as 10 ng/ml (6,23). Solid-Phase EGF Competition Assay. To quanti tate EGF competition in the HPLC eluates, a rapid solid-phase competitive binding assay suited for screening a large number of samples was developed and is discussed in detail elsewhere (12). In brief, receptor-rich A-431 (7) cell membrane fragments in 100 nI Dulbecco's phosphate-buffered saline, pH 7.2, were dried overnight at 37°onto 96-well polyvinyl chloride plates (Dynatech Laboratories, Inc., Arlington, VA), 2.5 /¿gmembrane protein per well. Receptor grade mouse EGF (Collaborative Research, Inc., Waltham, MA), was further purified to homogeneity over HPLC as described (2, 12,14), radtolabeted with 125I(Amersham/Searie Corp., Chicago, IL) as described (8), and used as the '"l-labeled binding component. HPLCpurified unlabeied mouse EGF was also used for deriving standard inhibition curves. Binding was initiated by the addition of 100 ¡Abinding buffer (6) [Dulbecco's minimum essential medium containing bovine serum albumin (1 mg/ml) and 50 mw 2|bis(2-hydroxyethyl)amino|ethanesulfonic acid adjusted to pH 6.8], containing 125l-labeled EGF (4 ng/ ml) with or without an aliquot of the sample to be tested. After a 1-hr incubation at 23°,the contents of the wells were aspirated, and the wells were washed 4 to 5 times with binding buffer (150 ^l). Finally, the wells were cut out of the plate with a hot wire, deposited into test tubes, and the amount of 125Mabeled EGF specifically bound was determined. EGFcompeting activity was quantitated as ng equivalents of EGF by com parison to a standard inhibition curve. Other Growth Factors. Sarcoma growth factor from Moioney sarcoma virus-transformed 3T3 cells (clone 3B11) was prepared and partially purified according to the methods of DeLarco and Todaro (6). Chemically homogeneous, recombinant HEGF was a generous gift from Chiron Research Laboratories (Emeryville, CA). RESULTS Previous studies revealed that urine from cancer patients contained a unique soft-agar colony-promoting activity that had a molecular weight of approximately 30,000 (23,27). In addition, elevated levels of a M, 6,000 to 8,000 fraction with colony- 3614 50 60 70 FRACTION 80 NUMBER Chart 1. Comparison of soft-agar growth-promoting activity and EGF-competing activity in HPLC fractions. Portions retained by ultratiltration of 10 mg of acidified urines were chromatographed as described in "Materials and Methods." Top, soft-agar colonies containing 8 to 20 cells/colony in 4 low-power microscope fields; bottom, EGF-competition represented as ng equivalents of EGF activity. •, 50-year-old male patient with renal cell carcinoma; O, 38-year-old male; , MeCN gradient. The tetters A to E designate each of the individual EGF-related TGFs resolved by HPLC. Gradient elution over a period of 2 hr at a flow rate of 1 ml/min, at room temperature, from 0 to 60% MeCN (Burdick and Jackson Labo ratories, Inc.) in 0.05% trifluoroacetic acid (Pierce Chemical Co.), adjusted to pH 2.5 with ammonium hydroxide, was used to develop the chromatogram after an initial isocratic elution for 5 min with 0.05% trifluoroacetic acid. One-mi fractions were collected. CANCER RESEARCH VOL. 44 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1984 American Association for Cancer Research. HPLC of TGF in Human Urine eluted at 32% MeCN, or of the activity (TGFD) which eluted at 37% MeCN, whereas these activities were observed at high levels in the cancer patient urine. Furthermore, the activity des ignated TGFE (38% MeCN) in the control urine appeared to be below the limits of detection in the patient urine. The levels of TGFB and TGFC had similar ranges in the urine of patients and normal controls. The observation that all of the above soft-agar colony-promoting activities were associated with EGF-competing activity and formed small-to-moderate-sized colonies in the ab sence of EGF conforms to the definition of a-TGFs, as previously described (18, 20,26). The foregoing suggested a potential difference between TGF activity profiles of cancer patients and normal individuals with respect to the relative amounts of TGFA, TGFD, and TGFE. To determine whether such differences were consistent, we per formed similar HPLC analyses on a series of urines from cancer patients and normal controls. Table 1 compares levels of colonyforming and EGF-competing activity for the above-mentioned TGFs in urine from 2 pairs of age- and sex-matched normal donors and cancer patients. Soft-agar growth-promoting and EGF-competing activity were concomitantly elevated for TGFA and TGFD in cancer patients but depressed in normal controls, whereas the converse was observed for TGFE. It may also be noteworthy that although there were similar levels of EGFcompeting activity for TGFC from normal male and female con trols, the levels of soft-agar growth-promoting activities showed a wider distribution. Also, the male urines demonstrated more TGFE than did female urines when tested by the radioreceptor assay but less TGFE when tested in the soft-agar assay. This may be due in part to additional factors which are present in female urine, as observed by Twardzik et al. (27), which could contribute to the colony-forming activity present in those frac tions. Because there are often large variations in the absolute level of growth-promoting activity in individual urines, and because the soft-agar bioassay often demonstrates day-to-day variation, comparisons between specimens were facilitated by determining the TGFA/TGFE ratio for the number of soft agar colonies and for ng equivalents of EGF-competing activity recovered from reverse-phase HPLC (Chart 2). The TGFA/TGFE ratio was <0.5 in all normal controls and >0.5 in all cancer patients for both soft-agar colony promotion and EGF competition. In order to determine the relationship of the above EGF-related TGFs to the tumor-associated M, 30,000 TGF described previ ously (23), high-molecular-weight TGF (M, 30,000) was isolated by gel filtration from the urine of a patient with breast carcinoma and then applied to reverse-phase HPLC. Chart 3 demonstrates that the Mr 30,000 TGF activity eluted with the same retention as TGFA (32% MeCN). Since normal control urine had been shown previously by gel filtration to contain little or no M, 30,000 TGF (23) and also lacked significant levels of TGFA activity by reverse-phase HPLC, we tentatively concluded that TGFA is probably equivalent to high-molecular-weight (approximately, M, 30,000) tumor-associated growth factor in urine. However, in order to further correlate the M, 30,000 TGF with TGFA, the latter was isolated from reverse-phase HPLC and was promptly rechromatographed over Bio-Gel P-30. The result of that exper iment, shown in Chart 4, reveals the presence of 2 molecular weight species, one of approximately M, 25,000 to 30,000 and one of approximately M, 6,000. Both demonstrated EGF-com peting activity and soft-agar colony-forming activity. It was there fore of further interest to determine whether the M, 6,000 TGF had arisen from the 30K TGF. Accordingly, a M, 30,000 TGF pool was isolated by gel filtration and, following storage for 4 days at 4°in the column eluant (1 M acetic acid), was rechro matographed by gel filtration over Bio-Gel P-30. The results Cancer Patients 7 * Normal Controls O u, 6 u_ p H 5 LL CD 4 i" = 2- EGr COMPETITION COLONY NUMBER Chart 2. Comparisons of ratios of TGFA/TGFE for normal controls and cancer patients. Values are for numbers of soft-agar colonies and for ng equivalents of EGF-competing activity. Table1 Comparison of growth factor activity in urine from patients and normal subjects Urines containing 10 mg of protein were concentrated to 500 n\ and applied to reverse-phase HPLC. Each fraction was analyzed for stimulation and growth of NRK cells in soft agar and for EGF competition on membranes of receptor-rich A-431 cells (see "Materials and Methods" for details). Equivalents of EGF in ng were calculated by means of an EGF competition curve using HPLC-purified mouse submaxillary EGF as standard. Values represent activity in peak fractions of the respective TGFs identified in Chart 1. No. of colonies of NRK cells in soft agar/starting protein analyzed 10 mg of urinary Donor31-yr-old mate with colon car (88)580 (144)0 (11)388 cinoma36-yr-old normal male (28) (66) (0) (50) 41-yr-old female with breast 930 (230)16 240 (40)380(80)TGFc270 220 (280)185 1100(1150)0 (10)244(220) 0 carcinoma 49-yr-old normal femaleTGFA300(155)"5 (25)TGFB110(22)154(22) (56)TGFo275 (0)TGFE0 " Numbers in parentheses, ng equivalents of EGF-competing activity per liter of urine AUGUST 1984 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1984 American Association for Cancer Research. 3615 E. S. Kimball et al. 40 R o LU 20 O o o 60 50 70 FRACTION rately chromatographed under the same conditions as was the urinary TGF (Chart 6). The tissue culture-derived sarcoma growth factor activity eluted earlier in the gradient, approximately 26% MeCN, and hence is less hydrophobic than is urine-derived TGF. Also, as shown, the HEGF eluted with the same retention time as did TGFc (see Chart 1) and we tentatively conclude that some, if not all, of the EGF-competing activity in that region of the chromatogram may be due to EGF. Since substantial soft-agar colony-forming activity was con sistently observed with TGFC>and because in our experience, at doses «10 mg/ml, neither mouse EGF nor HEGF intrinsically caused the formation of colonies by NRK cells, it seemed likely that some of the activity might be due to the presence of nonEGF-related TGF. Therefore, in order to further characterize the 80 NUMBER 150-1 Chart 3. Reverse-phase HPLC of tumor-associated high-molecular-weight TGF. Reverse-phase HPLC of M, 30,000 activity isolated by Bio-Gel P-30 chromatography, as described by Sherwin et al. (23). from an acid extract of urine from a female patient with breast carcinoma. Soft-agar growth-promoting activity (•) represented as number of colonies containing 8 to 20 cells in 4 low-power microscope fields. EGF-competing activity (O) represented as percentage of inhibition of binding of 18SI-EGF. , MeCN gradient used, as described in "Materials and Methods." 120- 90- 36-1 30K 36 25K 14K 8 32- 60-1 o 28 Z 28- 30- O — 24- I •»u. oUJ V) Ul 20- 20 - 8 16 o g 12- 20 8 20 24 28 32 36 FRACTION 40 44 52 Chart 4. Rechromatography of TGFA by gel filtration. TGFA, isolated after HPLC, was applied to a Bio-Gel P-30 column (2 x 40 cm) equilibrated in 1 M acetic acid and run at a flow rate of 0.2 ml/min. Two-mi fractions were taken, lyophilized, and then tested for colony-promoting activity (•) and EGF-competing activity (O). revealed that the Mr 30,000 TGF had been converted exclusively to a M, 6,000 TGF (Chart 5). These results suggest that there is a correlation between the M, 30,000 tumor-associated TGF and TGFA and further suggests that the M, 6,000 urinary TGF is at least in part derived from the M, 30,000 urinary TGF. Two other issues which were of concern were how the elution from HPLC of urinary TGF differed from that of TGF secreted in vitro by tumor cells and which of the various urinary EGF-related TGF reverse-phase fractions (TGFA-E) might actually contain urogastrone (HEGF). Accordingly, a sarcoma growth factor prep aration (6) and homogeneous recombinant HEGF were sepa- 3616 36 40 44 48 52 100-1 ^ NUMBER 32 Chart 5. Rechromatography of M, 30,000 TGF by gel filtration. M, 30,000 TGF, isolated by Bio-Gel P-30 chromatography, was rechromatographed using the same gel filtration column and conditions as in Chart 4. Samples were tested only for colony-promoting activity in soft agar. 90- fc? -4 28 FRACTIONNUMBER 12 t O I 8- 24 é 80- m u. 70- 7 60 in 0 son O Z 40- Z 20- o 10-ootÃ-50 60 70 80 FRACTION NUMBER Charte. Reverse-phase HPLC of sarcoma growth factor (SGF) and HEGF. Partially purified sarcoma growth factor and highly purified HEGF were individually applied to C-18 reverse phase and chromatographed using the same conditions as those used , MeCN for gradient urine concentrates used; |, elution (seeposition "Materials of TGFA. and Methods" and Chart 1). CANCER RESEARCH VOL. 44 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1984 American Association for Cancer Research. HPLC of TGF in Human Urine urinary TGF activities separated by HPLC and to look for other possible differences between cancer patient and normal urines, assays were performed for non-EGF-related 0-TGF-like activity. Accordingly, suboptimal amounts (2 ng) of HPLC-purified mouse EGF was added to urinary HPLC fractions in a soft-agar colony profiles between normal controls and cancer patients (not shown) could be discerned. assay. Results of a representative HPLC experiment with a urine sample taken from a normal female control are in Chart 7. In the absence of exogenous EGF, most colonies contained only 8 to 20 cells and coeluted with EGF-competing activity (a-TGF activ ity). The addition of purified mouse EGF (2 ng/ml) to the cultures enhanced the numbers of colonies for all fractions that contained EGF-competing activity and increased the size of those colonies to greater than 40 cells. Mouse EGF alone caused the formation of only low numbers of small colonies (<6 cells). In addition, when in the presence of added mouse EGF large colonies were found to be promoted by certain HPLC fractions (approximately, Fractions 65 and 91), eluting as broad, heterogeneous peaks with 30 and 43% MeCN. These fractions had shown no EGF receptor-binding activity and no colony-forming activity in the absence of EGF supplements. Hence, these fractions probably contain /3-TGF-like activity, as described previously (1, 18, 20). In contrast to the distinctions in a-TGF-like profiles between normal and cancer urine, no differences in /3-TGF-like activity Human urine has previously been shown to be a source of both HEGF (4, 9), which has limited soft-agar colony-promoting activity (6,23) and EGF-related transforming growth factor activ ity (23, 27). Gel filtration of acid extracts of urine showed that normal donors expressed low-molecular-weight EGF-related o LU < OC 2 C3 O 50 60 70 80 FRACTION NUMBER 90 ;co Chart 7. HPLC profilo of EGF-dependent and EGF-independent activity in urine from a normal female. Top, soft-agar colonies per 3 mg urine protein. •,colonies formed in the absence of EGF (EGF independent). Colony size was 8 to 20 cells/ colony. O, augmentation of EGF-independent colony formation activity and dem onstration of EGF-dependent colony-forming activity. Soft-agar colonies per 3 mg urine protein formed in the presence of 2 ng of supplemental mouse EGF. Colony size was typically »40cells/colony. Bottom, EGF-competing activity (O) per 3 mg of urine protein expressed as ng equivalents of EGF. DISCUSSION TGF activity (M, 6,000 to 8,000) and that the majority of cancer patients demonstrated an additional high-molecular-weight EGFrelated TGF activity (M, 30,000 to 35,000) (23, 27). The present study was undertaken to further analyze urinary TGF activity. We now report that reverse-phase HPLC analyses of acid extracts of urine from normal donors and cancer patients re vealed the presence of a total of 5 EGF-related growth factors with soft-agar colony-promoting activity and 2 non-EGF related factors which had colony stimulating activity only in the presence of added EGF. Of the 5 EGF-related activities observed, one (TGFA) was elevated in the urine from cancer patients and correlated with previously reported M, 30,000 to 35,000 tumorassociated TGF. A second (TGFD) occuring mainly in the urine from cancer patients, and a third, (TGFE), found at high levels only in normal control urines, were also identified. The relative proportions of the various a-TGF-like activities observed within each specimen tested were in general the same when tested for either soft-agar growth-promoting activity or EGF-competing activity. However, the EGF competition assay is a rapid, 1-day radioreceptor assay, vis-à -vis the soft-agar bioassay which requires 7 to 14 days. Moreover, the solid-phase EGF assay is as effective and sensitive as the soft-agar bioassay for distinguishing the HPLC-separated TGFs. Thus, we were able to demonstrate that Ultrafiltration, reverse-phase HPLC, and solid-phase EGF-competition assay techniques could be com bined to provide a rapid, convient procedure for monitoring EGFrelated growth factor activity expression in the urine. Using this technology, we were able to discriminate between urinary EGFrelated TGFs produced by cancer patients and normal controls. The soft-agar colony formation assay warrants further com ment. The colonies formed by the NRK cells were reported originally to consist of greater than 20 cells (5, 6) when sarcoma growth factor was used as a colony promoter. Other investiga tors report that purified EGF-related TGF (a-TGF) promotes formation of only very small colonies (18, 20). Similarly, there have been reports that EGF does not promote colonies (5, 6) or promotes large colonies (11,18). These differences may, in part, be due to differences in growth factor purity but may also reflect qualitative differences in the colonies which resulted from the different growth factors examined. Also, the NRK cell line has an increased sensitivity to growth factors and heightened sus ceptibility to spontaneous transformation when cells from higher passages are used (11). In the studies with human urine-derived growth factor activity, NRK cells were taken at passages 17 to 19 for soft-agar assays. Under these conditions, most of the colonies formed consisted of approximately 20 cells after a 7- to 14-day incubation period. Purified mouse EGF and HEGF caused the formation of few colonies, if any, containing no greater than 6 cells (23) when the EGF alone was used at concentrations as high as 10 ng/ml. In AUGUST 1984 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1984 American Association for Cancer Research. 3617 E. S. Kimball et al. the present study, an EGF supplement (2 ng/ml) was able to enhance the size and number of colonies promoted by some HPLC fractions that had EGF-competing activity and, in addition, was able to promote colony formation by HPLC fractions which otherwise had shown neither colony-forming nor EGF-competing activity. This is a novel discovery, since EGF-dependent growthpromoting activity in urine has not been described. Because there are reports that a-TGF colony-forming activity is not en hanced by EGF supplements (18, 20), this result suggests that suboptimal amounts of non-EGF-related TGF (/3-TGF) may be a contaminant in those a-TGF fractions examined and is consistent with the demonstration that 3 orders of magnitude less EGF than a-TGF is required to augment the promotion of /3-TGF colony formation (18,20). Further purification of the components in urine with growth promoting activity is required to further resolve this issue. Accordingly, we have purified recently a M, 6,000 EGFrelated TGF from melanoma patient urine which retained EGFcompeting activity and EGF-independent soft-agar growth-pro moting activity, and which upon isoelectric focusing revealed a single peak that was distinct from the isoelectric point of urogastrone.3 The presence of high levels of tumor-associated TGFA after HPLC purification of urine from cancer patients is in agreement with earlier observations by Sherwin ef a/. (23) of a unique M, 30,000 to 35,000 TGF activity found after gel filtration to be predominantly in urine from cancer patients. Our results indicate that the M, 30,000 TGF and TGFA are equivalent and that the M, 30,000 TGF wiill also convert to an active M, 6,000 TGF under conditions which have been reported not to promote conversion of high-molecular-weight HEGF (10). In addition, tumor-associ ated TGFA did not elute on reverse phase at the same retention as did HEGF. Thus, TGFA and M, 30,000 TGF are not HEGF, although the possibility exists that they may be either precursors or consist of altered forms of EGF. Finally, we observed that these urinary TGFs appear to be chemically distinct from tissue culture-derived TGF by displaying increased hydrophobic character. If the urinary tumor-associated TGF is, in fact, a tumor-derived substance and not the result of a host response, its increased hydrophobicity over that of a tissue-culture-derived TGF suggests that it may have undergone chemical alteration in vivo. This was shown to be the case in another study involving urine from tumor-bearing mice.4 The dramatically elevated levels of TGFA and TGF0, which were found exclusively in the urine from cancer patients could be the result of changes in normal TGF metabolism. Thus, the high-molecular-weight tumor-associated TGF, corresponding to TGFA, may be present in the urine from cancer patients because it is not as efficiently metabolized as it is in normal controls. This interpretation is based on analogy to the conversion of highmolecular-weight EGF to low-molecular-weight EGF (10, 25) and is consistent with the demonstration that partially purified highmolecular-weight TGF (M, 30,000) will gradually convert to lowmolecular-weight TGF (Mr 6,000). We are examining currently the chemistry of Mr 30,000 TGF conversion to M, 6,000 TGF. Immunological and biochemical comparisons to HEGF have also been undertaken to further resolve this aspect of our studies, 3M. K. Kim, T. C. Warren, and E. S. Kimball, manuscript in preparation. 4D R. Twardzik, S. Sherwin, E. S. Kimball, J. R. Ranchalis,and G. J. Todaro. A comparison of growth factors functionally related to EGF in the urine of normal and tumor bearing mice, submitted for publication. 3618 and we are in the process of completely characterizing these various TGFs in order to understand the biochemical relation ships which exist between them, to compare them to TGFs produced in vitro, and to ultimately understand their biological function. Finally, the observation that urine from cancer patients contains TGF activities which, via reverse-phase HPLC and a solid-phase EGF-competitive binding assay, can be distinguished from TGF activity found in normal controls further supports the possibility that TGF activity may be a clinically useful biology marker for certain types of cancer. REFERENCES 1. Anzano, M. A., Roberts, A. B., Meyers, C. A., Komoriya, A., Lamb, L. C., Smith, J. M., and Sporn, M. B. Synergistic interaction of two classes of transforming growth factors from murine sarcoma cells. Cancer Res., 42: 4776-4778,1982. 2. Burgess, A. 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AUGUST 1984 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1984 American Association for Cancer Research. 3619 Distinct High-Performance Liquid Chromatography Pattern of Transforming Growth Factor Activity in Urine of Cancer Patients as Compared with That of Normal Individuals Edward S. Kimball, William H. Bohn, Karen D. Cockley, et al. Cancer Res 1984;44:3613-3619. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/44/8/3613 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1984 American Association for Cancer Research.