Enzyme Activities and Isozyme Patterns in Human Lung Tumors1

[CANCER RESEARCH 44, 1058-1062, March 1984] Enzyme Activities and Isozyme Patterns in Human Lung Tumors1

Doris Balinsky,2 Olga Greengard, Eftihia Cayanis, and Jonathan F. Head

Department of Biochemistry, Iowa State University, Ames, Iowa 50011 [D. B.], and Departments of Pediatrics (0. G., E. C., J. F. H.] and Pharmacology [O. G.] Mount Sinai School of Medicine of the City of New York, New York, New York 10029

ABSTRACT in the proportions of LDH, HK, and PK variants in hepatocellular carcinomas (1.10) and, as demonstrated recently, of LDH, MDH, The Â¡sozyme patterns and activities of six enzymes were and PK isozymes in breast cÂ£,ncers(3). The present study of determined in surgical biopsy samples of lung tumors and non- these and additional parameters in human pulmonary tissues neoplastic pulmonary areas. Fetal lungs were also examined. No provides the first indication of consistent qualitative alterations tissue differences were found in the isozyme pattern of hexoki- of some enzymes, namely, of the pattern of LDH and MDH nase or alkaline phosphatase; small differences in pyruvate ki- isozymes. One of the quantitative indicators of neoplasia now nase isozyme proportions were observed. The tumors exhibited identified, Â«-GPD,was at diminished levels in each tumor. LDH, significant deviations with respect to the lactate dehydrogenase MDH, PK, and HK exhibited the opposite behavior, a concerted (LDH) and malate dehydrogenase (MDH) isozyme patterns. De elevation of their mean activities above those in nonneoplastic spite the diversity of cell types, the proportions of the M-subunit lung. Thus, an increase in glycolytic potential accompanies the of LDH in each tumor and that of the mitochondrial isozyme of enrichment of human pulmonary neoplasms with enzymes of MDH in all but one tumor were higher than in control samples DNA, RNA, and amino acid synthesis (8, 9). A preliminary report from the same lung. In contrast, the normal fetal lung showed a of some of this work has appeared (2). higher LDH-H proportion than did adult lung and a mature MDH isozyme pattern. The a-glycerophosphate dehydrogenase and adenylate kinase activities of the tumors were about one-tenth MATERIALS AND METHODS and one-fourth, respectively, of those of nonneoplastic adult Tissues. Adult pulmonary samples were from surgically resected lung. These lower activities (evident also in normal fetal lung) were accompanied by 3- to 5-fold increases in the LDH, MDH, specimens of patients undergoing lung surgery at the Mount Sinai Medical Center, New York, NY. The 15 pulmonary tumors comprised 7 pyruvate kinase, and hexokinase activities of the tumors; fetal adenocarcinomas (1 poorly, 5 moderately, and 1 well differentiated), 2 lungs had lesser increases (2- to 3-fold) for the first 3 enzymes. poorly differentiated large cell carcinomas, 3 moderately differentiated The common features of tumors with different cell types and squamous cell carcinomas, 1 poorly differentiated fibrosarcoma, 1 well- histological grade identified here point to several enzymes the differentiated mesothelioma (epithelial type), and one metastatic, well- differentiated colonie adenocarcinoma. Samples from the subjects' pul quantitation or isozyme analysis of which may be of practical monary parenchyma without apparent neoplastic involvement were also use in distinguishing cancerous from nonneoplastic human lung analyzed (when available) and will be referred to as "lung" or "adult lung." samples. A combination of different indicators, such as opposite changes in LDH and Â«-glycerophosphate dehydrogenase activ Normal lung samples from fetuses, obtained by the courtesy of Dr. ity, coupled with elevated proportions of LDH-M, may be used Kerenyi (Department of Obstetrics and Gynecology, Mount Sinai Medical Center) were from cases of elective abortions in the 9th to 15th week of to diagnose neoplasia most reliably. gestation. A portion of each tissue sample was preserved for histopath- ological examination. The remainder was usually divided into 2 portions, INTRODUCTION one of which was subjected to immediate biochemical analysis in New York, while the other was frozen at -20Â° and shipped for biochemical Our investigations in the past few years have focused on an analysis at Iowa State University, Ames, IA. examination of enzyme activities and isozyme forms occurring Enzyme Assays. For measurement of the activity of LDH, MDH, PK, in human tumors of the liver (1, 10), breast (3, 4), lung (6, 8, 9), and alkaline phosphatase, frozen tissue was homogenized in 9 volumes of 10 RIM Tris-HCI buffer, pH 7.4:0.1 mw dithiothreitol and centrifuged and colon (12). In pulmonary tissues, isozyme studies of carbo hydrate-metabolizing enzymes were restricted to AK,3 which for 30 min at 12,000 x g at 4Â°in an International refrigerated centrifuge. LDH, MDH, and PK were assayed as described previously (1), except exhibited a consistently lower activity in tumors than in the that Tris-HCI buffer was used, and 0.1 M KCI was added to the PK assay control lung, but its isozyme pattern remained normal (6). In mixture. Alkaline phosphatase was assayed by published methods (7, other human organs, more extensive isozyme analysis revealed 11). A fresh, nonfrozen portion of the same tissue was homogenized (in several cancer-associated abnormalities. Noteworthy among 0.14 M KCI) within 1 hr after resection, and the cytosol (obtained by these were the concomitant deviations (from the cognate tissue) centrifugation at 100,000 x g for 35 min) was assayed immediately for HK (1), AK (6), Â«-GPD(1), TK (19), UK (13), phosphoserine phosphatase 1Supported by National Cancer Institute Contract N01 -CB-84222 and Research (EC 3.1.3.3) (18), PK, and MDH (1). The results on the last 2 enzymes Grant CA-25005. were the same as those obtained (and shown under "Results") in aliquots 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: AK, ATP:adenosine 5'-phosphotransferase, EC of the tissues frozen and extracted as above. The enzyme activities are expressed as units per g of wet weight of tissue or per mg of protein, 2.7.1.20 (adenylate kinase); LDH, L-lactate:NAD oxktoreductase, EC 1.1.1.27 (lac where "unit" is defined as 1 Â¿imolof substrate converted/min. These tate dehydrogenase); MDH, L-malate:NAD oxidoreductase; EC 1.1.1.37 (malate dehydrogenase); HK, ATP:o-hexose phosphotransferase, EC 2.7.1.1 (hexokinase); activities, determined under optimal substrate and cofactor concentra PK, ATP:pyruvate phosphotransferase, EC 2.7.1.40 (pyruvate kinase); a-GPD, sn- glycerol-3-phosphate:NAD* 2-oxidoreductase, EC 1.1.1.8 (a-glycerophosphate de tions, were proportional to the amount of enzyme added. hydrogenase); TK, ATP:thymidine5'-phosphotransferase, EC 2.7.1.21 (thymidine Electrophoretic Methods. Prior to electrophoresis, the above-de kinase); UK, ATP:uridine 5'-phosphotransferase, EC 2.7.1.48 (undine kinase). scribed extracts of frozen tissue were centrifuged for 35 min in a Received July 27, 1983; accepted December 5, 1983. Beckman ultracentrifuge at 114,000 x g or 10 min in a Beckman Airfuge

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Isozymes in Lung Tumors at 197,000 x g. Approximately equal activities of enzyme were applied to each position on the strip. PK isozymes were separated on Cellogel in a 20-hr run, using the buffer system described by Strandholm ef al. * r-; CM CM (22). The other isozymes were separated on cellulose acetate strips by +1+1 running for 30 to 60 min in 10 rriM barbital:50 mw sodium barbital buffer, O) ID pH 8.6. All of the staining mixtures contained 0.85% agarose and 0.1 M Tris-HCI, pH 8.0. The stains for LDH, MDH, HK, and PK contained nitroblue tetrazolium (0.5 mg/ml) and phenazine methosulfate (0.04 mg/ ml); in the case of LDH and MDH, they contained, in addition, 0.4 mM â€¢*o> in o> NAD and 0.1 M lÃ¡clate (20) or 0.1 M malate, respectively. Ten ml of this +1+1 mixture were poured into a square disposable Falcon Petri dish, and the strip was laid on it in the dark at 37Â°until the desired staining intensity was attained. HK (17) and PK (1) were visualized by published methods. After the reaction had been stopp K! with 10% acetic acid, PK and HK isozymes were estimated visually. LDH and MDH isozymes were quan- as. titated by scanning the strips in a Beckman Model R-110 densitometer. Ã¶Ã¶ LDH is a tetramer consisting of M- (or A) and/or H- (or B) subunits, LDH, +1+1 being H4, LDH6 being M4, and the intermediate bands being hybrids of M and H. The percentage of the LDH-M subunit was calculated as follows:

% of LDH-M

_ 4(% of LDHs) + 3(% Of LDHÂ«)+ 2(% of LDH3) + % of LDH2 Â¡D T- O +1+1+1

RESULTS "Â»pv Electrophoresis of extracts of pulmonary tissues and tumors f T- CM revealed the presence in each specimen of all 5 forms of LDH. +1 +1 +1 Their quantitation showed, however, that the proportions of CM O CO LDH4 (M3H) and LDH5 (M4) in tumors were significantly higher than in the control lung, while LDHi, LDH2, and LDH3 were ss significantly lower (Table 1). The 4 fetal lungs studied (11th to CM Â»- CM 15th week of gestation) did not differ from the adult lung with +1 +1 +1

MARCH 1984 1059

tracts of each of these, as in the control lung samples, L- resis of neoplastic, normal, and fetal lung; HK II, which occurs in homoarginine was found to be a potent inhibitor of alkaline some tumors (16), was not detected in the pulmonary neoplasms phosphatase, and L-phenylalanine inhibited weakly (Table 1). of the present study. The AK isozyme pattern, as reported These data show that the Regan variant was not present in any previously (6), was the same in neoplastic and normal (fetal or of these tumors. adult) lung. Isozymes of PK and HK. PK consists of 3 major isozymic Enzyme Activities. The results of the quantitation of 6 en forms: L, which predominates in liver; M, which occurs in muscle zymes in adult, fetal, and neoplastic lung are listed in Table 2. and brain; and K, which predominates in most other tissues (15). The first 3 values in each line serve to compare these 3 types of Hybrids of K and M can form; these occur naturally in bovine tissues with respect to a given enzyme. Since the activities of kidney (5) and in human hepatoma (10) and brain (23). the different enzymes vary considerably, the behavior of the In this study, electrophoretic analysis of lung tumors showed different enzymes is best assessed from the activity quotients Â«4tobe the major isozyme in all but 1 of the 14 tumors examined. which, equal to 1.0 for nonneoplastic adult lung, reveal at a However, two-thirds (7 of 11) of the adult lung tissues also had glance the direction (and extent) of deviations from this reference K4 as the major band. A more anodal band, which could be a tissue. The tumors (see Table 2, AQT) showed similar elevations hybrid of 3K subunits with one M subunit or of 3K subunits and for LDH, MDH, PK, and HK; these, however, were accompanied a modified K""1subunit (14), was detectable in each specimen of by considerably lower activities of both Â«-GPDand AK. In fetal tumor or host lung. lungs (see Table 2, AQF), the extent of the deviations was smaller; Hexokinase HK I was the only variant found upon electropho- however, the direction of the deviations that were statistically significant (see superscripts) was the same as in the tumors in that LDH, MDH, and PK exhibited higher, and o-GPD and AK 60 lower, titers than did normal adult lung. Chart 2 shows the individual data points for LDH, MDH, PK, ~ 70 I 50 and Â«-GPDactivities in lung tumors (Ca) and in the corresponding 53 -Q "Ã•5 cognate lung tissue (N) from the same patient. To render the ? 60 ^40 charts comparable to those published previously by us and

6Â« others, the results are expressed in activity units per g, wet weight. One anomalous tumor value stands out; this specimen 50 Ã‹30 BÂ« had an abnormally low protein content (19 mg/g of wet weight of tissue, compared to the average of 46 Â±13 mg/g of tissue 40 20 N Ca N Ca for all tumors). Apart from this point, all of the tumor values for LDH and PK were outside of the normal range, and only one Chart 1. Isozyme proportions in normal and neoplastia lungs. A, LDH; 8, MDH. MDH and 2 Â«-GPDtumor values overlapped normal values. In A line connects the value for a tumor (Ca) with that for a control sample from the same lung (A/). every case, each tumor sample had higher enzyme activity (lower

Table 2 Enzyme activities in normal adult and fetal lung and in lung tumors Depending on the sample size, some or all of the enzymes were quantitated in the adult tissues (Columns 2 and 3) and in lungs from fetuses (Column 4). Activity quotients AQr and AQF were obtained by dividing the mean values in Columns 2 and 4, respectively, with the corresponding enzyme activity in Column 3. Significance of difference between mean values are shown by footnotes under AQr (tumor versus adult lung) and AQF (fetal versus adult lung). p values were calculated by Student's t test, using a 2-tail hypothesis.

activitiesLDH Enzyme

lung(15)c(15)(14)(12)(6)(11)0.001 lung27.90.9415.10.5724.50.930.32Â±5.2Â±0.15Â±4.4Â±0.08Â±8.2Â±0.17Â±0.08(4)(4)(4)(4)(11)AQ,'3.5"4.8"2.8^4.0"3.4'4.8"3.4'0.0097 Units/gUnits/mg proteinMDH of 0.22Â±

Units/gUnits/mg 7.9Â± proteinPK of 0.14Â±18.5Â±

Units/gUnits/mg proteinHK of 0.31Â±

Units/gUnits/mg 0.43Â± 3.2"(6)(12)0.012 Â±0.0025 proteinAK of 0.007Â±

Units/g 1.76Â± 0.38'0.11"0.15"AQ,0.94Â°2.5"0.76ei.(r1.13e2.9"0.64e0.97e0.25"0.48'0.73e0.1 proteina-GPDUnits/mg of 0.1320.076TumorÂ±11.2Â°Â±0.027Â± 0.3510.70Â±2.8Â±0.05Â±2.5Â±0.4Â±3.9Â±0.06Â±0.04Â±0.001Â±2.5Â±0.052Â±0.19(12)(12)(12)(12)Fetal0.1750.510.0015Â±1.0Â±0.029Â±0.12Â±0.004(6)(5)0.3/

Units/gUnits/mg 0.042Adult 3* of protein85.21.7755.81.273.81.541.730.0327.338 Â± 0.000824.40.3720.00.3021.60.320.50.0120.1Â±0.0035.8 ' AQr, activity quotient (tumor); ACt, activity quotient (fetal). 0 Mean Â±S.E. c Numbers in parentheses, number of tissues. " p < 0.001. 9 Not significant. 'p<0.05. gp< 0.005. â€¢p< u.uuÃ¶.

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found in many other tumors, including those of the breast (3). 300- 3.0 However, this is not diagnostic of cancer in general or for 200 - 1.0 carcinomas, since in rhabdomyosarcomas (21) and also human hepatocellular carcinomas (1) (in contrast to the present pulmo nary ones), the H-subunit was found to be at increased levels in 150 0.75 the tumor relative to the host tissue. The MDH isozyme pattern of human neoplasms had only been examined in breast tumors to date (3); deviations from the 100 0.50 .23 cognate tissue similar to those reported here were found. The mitochondrial isozyme of MDH was increased in the tumors not only relative to the soluble form but also in absolute terms (Chart 50 025 16), and this was accompanied by decreases in a-GPD activity (Table 2). Since both enzymes are components of shuttles transporting reducing equivalents between cytosol and mito N Ca N Ca N Ca N Ca chondria, these observations could indicate that neoplastic trans Chart 2. Enzyme activities in normal and neoplastic lungs. A, LDH; 8, MDH; C, formation in the lung leads to altered regulation of this shuttle PK; D, Â«-GRÃ’.A line connects the value for a tumor (Ca) with that for a control activity. The isozyme patterns of the 4 other enzymes studied sample from the same lung (A/). showed no significant change. The tumors appeared to contain the same variants of HK and alkaline phosphatase as did the in the case of a-GPD) than did the nonneoplastic specimen from normal lungs, and the differences, with respect to the PK iso- the same patient. a-GPD achieves as clear a discrimination (in zymes, did not appear to be significant (see "Results"). the opposite sense) as does LDH, in that the values for most In addition to a shift in isozyme pattern, there was an increase tumors are below the control range, and none failed to exhibit in lung tumors of the total activity (per g, wet weight, or per mg the deficiency when compared to the nonneoplastic sample from of protein) of LDH and MDH. Similar, approximately 3-fold in the host lung (Chart 2D). The mean values in Table 2 would creases were found in the mean activities of PK and HK (Table indicate that measurement of AK is less effective than is mea 2) and, as shown for a comparable spectrum of human lung surement of a-GPD in distinguishing a neoplasm from noncan- tumors, in that of aldolase (9). Previous studies of a diverse cerous lung in general. However, in confirmation of results on a group of human lung neoplasms revealed coordinated elevations larger number of equally diverse cases (9), each tumor had a in enzymes involved in the synthesis of nonessential amino acids lower AK activity than did the control lung sample from the same and in the utilization of different, preformed nucleosides (8, 9). individual. The results on 3 additional enzymes reported in that The concerted elevations in the activity of different glycolytic study were also confirmed here: the activities of TK, UK, and enzymes described in the present study indicate that a superior phosphoserine phosphatase in each tumor [11.7 Â±13.4 (S.D.) equipment for energy metabolism contributes to the capacity of milliunits/g, 50 Â±28 milliunits/g, and 248 Â±174 units/g, wet these tumors for nucleic acid synthesis and thus for growth. weight, of tissue, respectively] were always higher than in the The several enzymes of nucleic acid and amino acid metabo control samples of the lung [0.40 Â±0.12 milliunits/g, 5.17 Â±2.5 lism which were at higher concentrations in human fetal lung milliunits/g, and 55 Â±39 units/g, wet weight, of tissue, respec than in adult lung have invariably been found to be elevated in tively]. pulmonary neoplasms (8, 12). Similarly, in this study, the LDH, MDH, and PK activity of both fetal tissues and tumors was DISCUSSION elevated, while those enzymes which changed in the opposite direction during normal development (AK and a-GPD) were at a The series of lung tumor cases were unselected in that they diminished level in the tumors. Whether or not this phenomenon included any subject from whom, from the surgically resected extends to isozyme patterns is not clear from the 5 enzymes so material, 2 samples (judged by the surgeon as "tumor" and far studied from that point of view. The cold-labile activity relative "lung," respectively) of at least 500 mg each were made available to the cold-stable activity of pyrroline 5-carboxylate reducÃase to us. Interpretation of the biochemical finding awaited the his- (1-pyrroline 5-carboxylate: NAD+ oxidoreductase, EC 1.5.1.12) of topathological examination of portions of each sample preserved fetal lung did deviate from adult lung in the same direction as in for this purpose. This examination disclosed which were the lung lung tumors (12), whereas the proportions of LDH subunits did samples, showed that they were free of neoplastic involvement, not (Table 1). The question was not applicable to MDH, PK (see diagnosed the tumors as "cancer," and revealed that the 15 "Results"), or AK (6), since the proportions of their isozymes in tumors were of 6 different cellular origins, with an uneven rep fetal lung did not differ significantly from those in normal adult resentation of degrees of histological differentiation (see "Mate lung. rials and Methods"). Thus, the numbers of tumors in groups with Charts 1 and 2 show that there is considerable variation in the same descriptions were too small to determine whether the individual control values for all enzymes and isozymes shown. extent of any enzymic alteration (compared to normal lung) is or This differs from the relatively small, random individual variation is not dependent on the cell type or histological grade of the found for TK and UK (8). In the latter case, deviation from the tumor. established control value would clearly indicate the cancerous Despite the diversity of histogenesis, the tumors shared the nature of a given biopsy sample. In the case of LDH, MDH, PK, property of containing higher proportions of the M-subunit of and a-GPD, overlap of values for control and tumor tissues is LDH than did the nonneoplastic lung samples. Relatively high seen; however it is evident that comparison of malignant and levels of LDH5 and thus of the M-subunit of LDH have been control samples from the same patient clearly shows alteration

MARCH 1984 1061

D. Balinsky et al. of activity and isozyme proportion in the tumor sample, whereas 9. Greengard, O., Head, J. F., Goldberg, S. L., and Kirschner, P. A. Enzyme pathology and the histologiecharacterizationof lung tumors: the continuum of a particular malignant sample may not differ from the mean of quantitative biochemicalindices of neoplasticity.Cancer(Phila.),49:460-467, the control values for different individuals. Similar conclusions 1982. were drawn previously for AK (8). 10. Hammond, K. D., and Balinsky, D. Isozyme studies of several enzymes of carbohydrate metabolismin human adult and fetal tissues, tumor tissues, and To achieve maximally reliable distinction with a minimal number cell cultures. Cancer Res., 38:1323-1328,1978. of assays, it is considered best to use more than one type of 11. Hammond, K. D., Balinsky, D., Bersohn, I., and Jersky, J. J. Kinetic studies on alkalinephosphatasefrom liver of the baboon,Papioursinus. Int. J. Biochem., criterion (e.g., an isozyme difference) and one enzyme which 4:511-520, 1973. would have a higher activity, as well as one which would have a 12. Herzfeld.A., and Greengard,O. Enzymeactivities in humanfetal and neoplastic lower activity (e.g., LDH as well as a-GPD) in a cancerous relative tissues. Cancer (Phila.),46: 2047-2054,1980. 13. Herzfeld, A. H., and Raper, S. M. Uridinekinase activities in developing adult to the control nonneoplastic lung sample. and neoplastic tissues. Biochem. J., 782: 771-778,1979. 14. Ibsen, K. H., ChiÃ¹,R.,Park, H. R., Sanders, D. A., Roy, S., Garratt, K. N., and Mueller, M. K. Purification and properties of mouse pyruvate kinase K and M and of a modified K-subunit. Biochemistry, 20:1497-1506,1981. REFERENCES 15. Imamura,K., and Tanaka, T. Multimolecularforms of pyruvate kinase from rat and other mammaliantissues: I. Electrophoretic studies. J. Biochem.(Tokyo), 1. Balinsky, D., Cayanis. E., Geddes. E. W., and Bersohn, I. Activities and 77:1043-1051,1972. isoenzymepatterns of someenzymesof glucose metabolismin humanprimary 16. Kamel, R., and Schwarzfischer, F. Hexokinaseisozymes in human neoplastic malignanthepatoma. Cancer Res., 33: 249-255,1973. and fetal tissues: the existence of hexokinase II in malignant tumors and in 2. Balinsky, 0., Jenkins, P. S . Ratz, C. E., and Lewis, J. W. Isozymes in human placenta. Humangenetik,30: 181-185,1975. breast and lung tumors. Fed. Proc., 40:1777,1981. 17. Katzen, H. M., and Schimke, R. T. Multiple forms of hexokinase in the rat: 3. Balinsky,D., Ratz, C. E., and Lewis, J. W. Isozyme patterns of normal,benign, tissue distribution, age dependency, and properties. Proc. Nati. Acad. Sci. and malignanthuman breast tissues. Cancer Res., 43: 5895-5901,1983. U. S.A., 54:1218-1225,1965. 4. Balinsky, D., Ratz, C. E., and Lewis, J. W. Enzyme activities in normal, 18. Knox, W. E., Herzfeld, A., and Hudson, J. Phosphoserinephosphatasedistri dysplastic, and cancerous human breast tissues. J. Nati. Cancer Inst., 72: bution in normal and neoplastic rat tissues. Arch. Biochem. Biophys., 132: 217-224,1984. 397-403,1969. 5. Cardenas, J. M., Dyson, R. 0., and Strandholm, J. J. Bovine and chicken 19. Machovich. R., and Greengard, 0. Thymidine kinase in rat tissues during pyruvate kinase isozymes. Intraspecies and Â¡nterspecieshybrids.In: C. L. growth and differentiation. Btochim. Biophys. Acta, 286: 375-381,1972. Marken (ed.), Isozymes, Vol. 1, pp. 523-541. New York: Academic Press, 20. Raabo, E. Colorimetrie method for determining lactic dehydrogenase isoen- Inc., 1975. zymes separated by paper electrophoresis. Scand. J. Clin. Lab. Invest., 75: 6. Cayanis, E., Greengard, O., and Iliescu, C. Adenylate kinase isoenzyme 405-414,1963. patterns in normal and neoplastic human lung and in various adults as 21. Schapira,F., Micheau.C., andJunien,C. Foetalenzymepattern of two alveolar compared to fetal rat tissues. Enzyme (Basel),25: 396-399,1980. rhabdomyosarcomas.Rev. Eur. Etud. Clin. Btol., 77: 896-899,1972. 7. Fishman,W. H. Oncodevelopmentalisoenzymes. In: P. L. Wolf (ed.), Tumor- 22. Strandholm, J. J., Dyson, R. D., and Cardenas,J. M. Bovine pyruvate kinase Associated Markers, pp. 73-80. New York: Masson Publishing,Inc.. 1979. isozymesand hybrid isozymes.Arch. Biochem.Biophys., 773:125-131,1976. 8. Greengard, O., Head, J. F., and Goldberg, S. L. Uridine kinase, adenylate 23. van Veeten,C. W. M., Verbiest, H., Vlug, A. M. C., Rijksen, G., and Staat, G. kinase, and guanase in human lung tumors. Cancer Res., 40: 2295-2299, E. J. Isozymes of pyruvate kinase from human brain, menmgiomas. and 1980. malignantgltomas.Cancer Res., 38: 4681-4687, 1978.

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Doris Balinsky, Olga Greengard, Eftihia Cayanis, et al.

Cancer Res 1984;44:1058-1062.

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