tant of KiMSV is thermolabile relative to that from wild type KiMSV-infected cells. The Kirsten murine sarcoma virus is a highly oncogenic helper-dependent type ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No.20,Issue of October 25,pp. 10583-10591, 1981 Printed in U.S. A .
LDHk, a Uniquely Regulated Cryptic Lactate Dehydrogenase Associated with Transformation by the Kirsten Sarcoma Virus* (Received for publication, January 15, 1981)
Garth R. Anderson+, WilliamP. Kovacik, Jr.+,and KeithR. Marotti8 From the Department of Microbiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
A novel isozymeof lactate dehydrogenaseis detected a 2-kilobase regionat the5’ end of the HaMSV genome. This includes a terminal 0.69-kilobase region with extensive hoinvarious cells transformedby theKirstenmurine sarcoma virus (KiMSV). This isozyme,designated mology to both the Kirsten sarcoma virus and the 30 S rat LDHk,is strongly inhibited by physiological concentraRNA species, a 0.35-kilobase region with little homology to tions of oxygen, in an apparently cooperative fashion. either species, and three more regions of varied homology LDHk is inhibited by guanosine triphosphate and re(Chien et al., 1979). Ellis et al. (1980) have presented data LDHk is suggesting that the rat 30 S RNA is not relevant tothe lated compounds, in a noncompetitive fashion. found withboth35,000-and22,000-daltonsubunits, transforming gene of HaMSV, based on a lack of homology although these probably cleave from a 57,000-dalton between a transfectible transforming DNA fragment and a precursor. single incomplete clone of the rat 30 S RNA. They propose In studiesutilizingatemperature-sensitivetransthe transforming activity of HaMSV resides in other, unique forming gene mutant of the Kirsten sarcoma virus, we find in vivo expression of LDHk is also temperature- sequence genetic information of rat origin. sensitive. In studies using either crude cell-free extracts In vitrotranslation of KiMSV viral RNA provided evidence or purifiedLDHk,we find the enzyme from cells infected for a 50,000-daltonpolypeptide gene product of KiMSV (Shih et al., 1978). In vitro translation and immunological studies with a temperature-sensitive transforming gene mufrom wild with sera from tumor-bearing rats have identified a 21,000tant of KiMSV is thermolabile relative to that dalton polypeptide as a HaMSV gene product (Shih et al., type KiMSV-infectedcells. 1978; Shih et al., 1979a).Cross-reacting p-21 material was seen in KiMSV-infected cells and immune precipitability of this p21 polypeptide was thermolabile in cells infected with a ts The Kirsten murine sarcoma virus is a highly oncogenic transforming gene mutant of KiMSV, ts 371. This would helper-dependent type C virus, with a genome containing type indicate the p-21 polypeptide is either a transforming gene C mouse leukemia virus sequences recombined with one or product of KiMSV or else it associates in an immune complex two sets of specific rat cell genetic information (Scolnick et with such a gene product (Shih et al., 1979b). GTP has been al., 1973; Anderson and Robbins, 1976; Chien et al., 1979; Ellis found bound in immune complexes containing the p-21 polyet al., 1980). Based on work with this and other avian and peptide (Scolnick et al., 1979) and GTP binding activity comurine sarcoma viruses, it is now clear that rat sequences of purifies with the p-21 polypeptide (Shih et al., 1980). The pthe KiMSV’ genome encode its transforming activity (Ste- 21 polypeptide appears to localize on or near the interior helin et al., 1976; Anderson et al., 1979; Anderson et al., 1979; surface of the plasma membrane (Willingham et al., 1980). Shih et al., 1979a, 1979b; Weiet al., 1980). Anaerobic stress massively induces uninfected rat cells to Most of the rat genetic information in KiMSV is homolo- express the rat30 S RNA (Anderson and Matovcik, 1977).We gous to and apparently derived from a 30 S RNA species have found appropriate treatment with cyanide ion also infound in normal rat cells (Anderson and Robbins, 1976; Chien duces this RNA.’ Immunological studies have identified a et al., 1979).This 30 S RNA appears to represent a number of common antigen in rat cells induced by anaerobic stress to closely related but not identical RNA species (Keshet et al., express the rat30 S RNA and in various non-rat cells contain1980). Cot curve analysis has indicated that there are around ing this same rat genetic information as a result of infection 30 copies of this DNA per haploid rat genome (Anderson and with the Kirsten sarcoma virus (Anderson et al., 1979;Marotti, Robbins, 1976) and thisDNA has properties suggesting it may 1979). This antigen, which is not seen in cells transformed by be related to transposons (Keshet and Shaul, 1981). Chang et other agents, is a 35,000-dalton polypeptide which co-purifies al. (1980) have used transfection studies to localize the trans- with lactate dehydrogenase activity (Anderson et al., 1979). forming region of the closely related Harvey sarcoma virus to We report here characterization of the KiMSV-associated * This work was supported by research grants from the National isozyme of lactate dehydrogenase, designated LDHk. LDHk, Science Foundation and the Muscular Dystrophy Association. The which is seen in Kirsten sarcoma virus-infected non-rat cells costs of publication of this article were defrayed in part by the and in anaerobically stressed rat cells, has novel structural payment of page charges. This article musttherefore be hereby marked ‘‘advertisement” in accordance with 18 U.S.C. Section 1734 and biochemical properties. Both in uiuo and as purified in uitro, this isozyme is found t o be thermolabile in temperaturesolely to indicate this fact. Present address, Departmentof Cell and Tumor Biology, Roswell sensitive transforming gene mutant KiMSV-infected cells, as Park Memorial Institute, Buffalo, NY 14263. compared to thatin wild type KiMSV-infected cells.
*
8 Present address, Department of Chemical Biology, The Rockefeller University, New York, NY 10021. ’ T h e abbreviations used are: KiMSV, Kirsten murine sarcoma virus; HaMSV, Harvey murine sarcoma virus; LDHk, KiMSV-associated isozyme of lactate dehydrogenase; KiMuLV, Kirsten murine leukemia virus; SDS, sodium dodecyl sulfate.
10583
EXPERIMENTALPROCEDURES
Cell Lines, Viruses, and Tissue Culture-The
* J. P. Scott
NRK rat cells were
andG . R. Anderson, manuscript in preparation.
LDHk in KiMSV Transformed Cells
10584
obtained from the laboratory of Stuart Aaronson, National Institutes of Health. Wild type and the temperature-sensitive transforming gene point mutant of KiMSV, ts-371, were generously supplied by Ed Scolnick, National Institutes of Health. Vero monkey cells were obtained from J . S. Youngner, University of Pittsburgh. Cells were grown on Falcon plastic culture dishes in the Dulbecco modification of Eagle's medium (Gibco). Cells were last fed 72 h before harvest. Preparation of Cell Extracts-Cells are rinsed with phosphatebuffered saline, scrapedfrom the culture dishes with a rubber policeman, and swollen in RSB (0.01 M Tris, pH 7.5, 0.01 M KC1, 0.0015 M MgClz,0.001 M dithiothreitol). Homogenization is achieved with 20 strokes of a Dounce homogenizer. Debris is removed by centrifugation for 5 min at 1,OOO X g and then anS-100 is prepared by centrifugation for 1 h at 100,OOO X g. Lactate Dehydrogenase Assay a n d Isozyme Gels-Total LDH assay, spectrophotometrically monitoring the conversion of NADH2 to NAD' in a pyruvate-utilizing reaction, is according to theprocedure of Schwartz and Bodansky (1966). Nondenaturing gel electrophoresis is used for isozyme analysis. Two slab gel systems are used. Normal polarity gels, which detect the standard M and H isozymes of LDH which migrate anodically, are run according to the procedure of Dietz and Lubrano (1967). This is a 5.5% gel in a Tris/glycine buffer, with the gel a t pH 8.9 and the electrophoresis buffer a t pH 8.3. Reverse polarity gels are used to isolate the apparent KiMSVspecific isozyme of LDH, LDHk, which has a positive charge even at pH 8.9. These gels are based on an imidazole/borate buffer system. These are 5.58 polyacrylamide gels photopolymerized with a riboflavin catalyst in the absence of persulfate. These gels contain 0.15 M potassium borate, pH 8.3. The upper reservoir buffer is composed of 0.08 M imidazole and 0.02 M boric acid, pH 8.9. The lower reservoir buffer is 0.1 M potassium borate, pH 8.3. Gels are run 16 h at a constant voltage of 220 V, with samples migrating toward the cathode. Cooling is provided by circulating ice water. In all cases, at thecompletion of the run, gels are stained specifically for lactate dehydrogenase activity by the nitro blue tetrazolium/ phenazine methosulfate procedure of Dietz and Lubrano (1967) supplemented with 5 mM NaCN and 2% glycerol to complex with the borate. Gels are fixed with 458 methanol/lO% acetic acid, then photographed or scanned with a tungsten filament recording densitometer (Quick Scan Jr., Helena Laboratories). The enzyme assay was determined to be essentially linear for at least the first 4 h. A 2-h incubation period was selected for standard assay conditions, to represent the initial rate of reaction. The gelstaining reaction was found to saturatewith the production of around 80 nmol of NADH*.Activity was computed for those assays producing less than 50 nmol of NADH,, where gel activity staining was linearly proportional to input protein. Purification of LDHk-The S-100 supernatant cell extract in RSB is concentrated by lyophilization and then chromatographed on Sephadex G-200. Fractions containing LDHactivity arethenfurther purified by affinity chromatography on Affi-Gel blue (Bio-Rad) and eluting with a linear salt gradient in NADH,, using procedures we have published (Anderson et al., 1979). LDHkis then furtherpurified 1.0 slab gels1.0 by nondenaturing gel electrophoresis on preparative using the imidazole/borate system described above. RESULTS
KiMSV Infection is Predominantly Associated with One LDH Isozyme-We have described evidence that transformation by the Kirsten sarcoma virus is associated with expression of a 35,000-daltonantigen which is detectable in a variety of Kirsten sarcoma virus transformed cells, but not in cells transformed by other agents (Anderson et al., 1979). This antigen co-purified with lactate dehydrogenase activity. We have now developed an imidazole/borate-based polyacrylamide gel system and assay procedures which effectively separate the KiMSV-specific isozyme of LDH from other LDH isozymes. As described in Table I, NRK cells transformed by the Kirsten murine sarcoma virus show around a 4-fold elevation of total lactate dehydrogenase activity, as assayed in aconventional spectrophotometric assay. However, when this is broken down into analysis of individual isozyme components, one specific isozyme of lactate dehydrogenase
was found to be affected in a most profound fashion, with around a 50-fold elevation in activity. This isozyme, designated LDHk, is highly basic and migrates toward the cathode in gels run at pH 8.9. (It should be noted that detection of this isozyme requires assay in the presence of cyanide or under a nitrogen atmosphere, as described in detailbelow). As detailed in Table I, in vivo expression of the isozyme LDHkis temperature-sensitive in cells infected with the ts transforming gene mutant of KiMSV, ts 371 (Shih et al., 1979a, 1979b).Infected cells grown at thepermissive temperature for transformation (32 "c)have LDHk levels elevated to anextent comparable to that seen with wild type KiMSV-infected cells,while cells infected with ts 371 and cultured at the nonpermissive temperature (39 "C) show no elevation ofLDHk over that low level detectable in uninfected rat cells. The use of KiMSV-infected rat cells offers an obvious drawback in that the KiMSV transforming gene is composed of rat sequences. Thus it might be considered advantageous to work with KiMSV-infected monkey or human cells in the hope that no background expression ofLDHk would occur. However, both primate lines also express trace levels of an LDHk, which essentially co-migrates with the ratenzyme, and responds similarly to assay under nitrogen (Anderson and Kovacik, 1981). LDHk from ts KiMSV-infected Cells is Thermolabile in Vitro-Reduced expression of LDHk in ts 371 KiMSV-infected cells cultured at 39 "C could reflect either 1)that some or all of the peptide constituents of LDHk are themselves thermolabile, indicating they are encoded by the ts KiMSV, or else 2) that LDHk is a hostenzyme expressed as a secondary effect of neoplastic transformation. If the fist case is true, the LDHk activity isolated from ts 371 KiMSV-infected cells grown at 32 "C should be thermolabile in uitro, whereas in the second, it should not. To evaluate these possibilities, we cultured NRK cells infected either with ts 371 KiMSV or wild type KiMSV at the permissive temperature (32 "C), where both were transformed. Cell-free extracts were prepared and then the LDHk was analyzed for its thermalstability (Fig. 1). TABLE I Lactate dehydrogenase in ts KiMSV-infected cells LDH activities, relative to uninfected cells Cells
Infected with
cultu;;d
LDH activity
Anodic Total"
EO-
LDHr'
zymesh
NRK 1.0 0.93 NRK 58.3 NRK 2.7KiMSV 4.0 3.70 80.2 NRK 3.9KiMSV 5.2 4.82 NRK ts371-KiMSVd 4.4 4.09 NRK ts371-KiMSV0.96
"C 32 39 32 39 32 39
IU/mg
1.010.3
1.1
0.6
68.2 3.3 1.0 1.1
0.8
" Total LDH activity was determined spectrophotometrically, by the procedure of Schwartz and Bodansky (1966). S-100 extracts of cells cultured at the indicated temperatures were assayed directly. Activity is relative to uninfected NRK cultured at 32 "C. * Anodic LDH isozymes were quantitated by running 15 pgof s100 protein on nondenaturing Tris/glycine gels, by the procedure of Dietz and Lubrano (1967). Gels were activity-stained and then scanned on an integrating densitometer. 'The isozyme LDHk was quantitated by running 15 pg of 5-100 protein on imidazole/borate gels, run toward the cathode. Gels were activity-stained in the presence of 5 mM NaCN and then scanned as in Footnote 6.
