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Detection of a novel lactate dehydrogenase isozyme and an apparent differentiation-associated shift in isozyme profile in hepatoma cell lines
CANCER LETTERS Cancer
Letters
87 (1994) 193-198
Detection of a novel lactate dehydrogenase isozyme and an apparent differentiation-associated shift in isozyme profile in hepatoma cell lines T.Z. Liu”, P.Y. Chen”, D.T.Y.
Chiua, J.S. Wei”, K.S.S. Changb,
K.H. Lin*b
‘School of Medical Technology. Chang Gung College of Medicine and Technology. Kwei-Shari.. Taoyuan. Taiwan, ROC bGraduate Institute of Clinical Medicine, Chang Gung College of Medicine and Technology, Kwlei-Shan. Taoyuan, Taiwan, ROC
Received
26 August
1994; revision received
I I October
1994; accepted 12 October
1994
Abstract
A hitherto unreported lactate dehydrogenase (LD) isoenzyme, which migrates electrophoretically to the relative position between LD, and LD, has been identified in the electropheratogram in 7 of 7 (lOO’%) cultured hepatoma cell lines with various degrees of differentiation and is thus given the name LD,_,. LD2_3 seems to be specific for hepatoma cells because this atypical isoenzyme can not be detected in other tumor cell lines. In addition, the hepatoma cell lines also show a distinct pattern of LD isoenzyme and the isoenzyme pattern varies with the degree of differentiation. Hence, the expression pattern of LD isoenzyme phenotypes may provide a good marker for the investigation of human hepatoma cell differentiation. Keywork
Hepatoma
cells; Differentiation;
Atypical
LD isozyme; Electrophoretic
1. Introduction
The control of cell differentiation remains one of the most challenging problems in biology. However, an experimental model for studying the differentiation of hepatocytes is still lacking at present. The fact that a variety of plasma protein or plasma membrane protein markers of hepatocytes have been well characterized [l-5] has led Chang et al. [6] to propose an approach for study* Corresponding +Ol
author, Tel.: +Oll
886 3 3288263;
I 886 3 3280174.
0304-3835/94/$07.00 @ 1994 Elsevier Science Ireland SSDI 0304-3835(94)03601-E
Fax:
patterns
ing human hepatoma differentiation based upon the expression patterns of plasma proteins. However, this technique, though specific, is quite laborious and lengthy because of the necessity to analyze at least 15 different plasma proteins of the culture medium. Furthermore, a variety of hepatocyte-specific gene expressions have been shown to be closely regulated during differentiation. Regulation of the expression of gene encoding proteins that are secreted by the liver is predominantly controlled at the transcriptional level, and is mediated through the action of a set of hepatocyte-specific trans-acting factors [7- IO].
Ltd. All rights reserved
194
T.Z. Liu et al. /Cancer
These hepatocyte transcription factors may serve as the markers for differentiation. However, their assays can be quite cumbersome. Thus, it is desirable to find an alternative marker which is simple and rapid to perform in order to study differentiation of hepatocytes. Lactate dehydrogenase (LD; EC 1.1.1.27) is a glycolytic enzyme present in various tissues. It is well documented that LD is coded for by three genes, giving rise to M, H, and X subunits, while the X subunits are produced in all tissues, although in various amounts. Five isoenzymes of LD have been reported: homotetramers M, (LDJ and H4 (LD,), and heterotetramers M3H (LD& MzH, (LD,) and MH3 (LD3. It is generally accepted that cells undergoing transformation can have concurrent alteration of gene expression leading to the change of isoenzyme patterns. As an example, the isoenzyme patterns of LD are altered in the serum and in affected tissues of patients with neoplasma [13-l 71. Furthermore, Liu [ 181 compared the LD isoenzyme patterns of cultured colonic and pancreatic tumor cells and found that they had a preponderence of M subunits, ranging from 60 to 100% and had either LD4 or LD, as the predominant isoenzyme. In addition, the majority of tumor cells tested had been shown to be either completely devoid of or deficient in LDI. These studies indicate that the expression of LD isoenzyme patterns seems to be changeable and inducible depending on the state of proliferation. Thus far, to the best of our knowledge, the relationship between the degree of differentiation and production of LD isoenzyme patterns in cultured hepatoma cell lines has not yet been delineated. Herein we report the data of our investigation dealing with this interesting problem.
Lett. 87 (1994) 193-189
2.2. Preparation of cell homogenates Cells cultured in dishes (100 mm diameter) were washed two times with isotonic PBS (pH 7.4), scraped from the dish and resuspended in 1 ml of PBS. Twenty microliters of 0.1 M dithiothreitol solution was added to the cell suspension before sonication for 20 s in a cold ice bucket. The cell homogenates were centrifuged at 100 000 x g for 30 min and the supemate was centrifuged again and separated for use in the electrophoretic studies. 2.3. LD isoenzyme quantitation LD isoenzymes of the supematant fractions were separated electrophoretically using Corning agarose universal electrophoresis film. To ensure proper assignment of the relative migrating position of LD isoenzymes, the first lane of each electrophoresis film plate contained normal human serum as the marker. Sample size of application depended on the protein contents of supernatant fractions. Approximately equal amounts, normalized by the protein content of each supernatant, was applied to the assigned position of the electrophoresis film plate. Electrophoresis was carried out in a Corning universal barbital buffer containing 0.035% EDTA at pH 8.6 for 45 min. After electrophoresis, 1 ml of AMP lactate solution in 20% (w/v) sucrose (pH 9.0) was evenly dispensed onto the agarose surface along the edge of a serological pipette. After incubation for 15 min, the agarose gel film was oven-dried (60°C) and the LD isoenzyme profile was scanned using a fluorescence densitometer (Corning Model 740). The percentage of the M subunit was calculated as described previously by Balinsky et al. [16]: % of M =
2. Materials and methods 4(% LD.J + 3(%LD,)+ 2. I. CeN lines procurement and culture procedure Hepatoma cell lines were kindly provided by C.P. Hu (Cell Bank of Medical Research Unit of VA Hospital, Taiwan). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 0.1 mM non-essential amino acids. The cultures were maintained at 37°C in humidified air with 5% coz.
2(%LD,)
+ (%LD2)
4
3. Results Table 1 summarizes the sources and istics of seven lines of HCC cells. All of cells were previously classified into gories of differentiation depending on
characterthese HCC three catetheir mor-
T.Z. Liu et al. /Cancer Table I Origin and characteristics Cell line
of human
Sex
Group I (well-differentiated) Hep G2 M M Hep 3B
hepatoma
black
Group II (intermediate-differentiated) PLCIPRFIS African black M F Chinese J5 Group III (poorly-di@erentiated) African black Mahlavu M Chinese proteins HA22T M SK-Hep- 1 M Unknown ‘Lin,
K. H. et al. (unpublished
phologies and plasma protein Fig. 1 shows types of HCC entiation. For
References
Morphologically similar to typical hepatocytes and secrete approximately 15 plasma proteins
I,19 1,19
Morphologically similar to elongated hepatoBytes and secrete about 9 plasma proteins
20
Morphologically similar to fibroblast-like tocyte and secret 0 to 6 plasma proteins
20 21 22
Well
Hep 38 PLC I Differentiation
Mahlavu 221 1
SK-Hepl
hepa-
data)
sertJm control
J-5
195
Characteristics
capabilities of expressing various markers. the LD isoenzyme patterns of seven cells with various degrees of differcomparative purposes, we also in-
Hep G2
193-189
cell lines used in this study
Race
Caucasian American
Lett. 87 (1994)
Poor
eluded four colonic tumor cells (SW-620, SW-480, HCT-8, HCT-48), two pancreatic tumor cells (CAPAN-1, UCVA-1) and two melanoma tumor cells (H2169 T, H2058). As indicated in Fig. 1, an atypical LD isoenzyme (named LD,_, in this report) migrates electrophoretically to the relative position between LD2 and LD3 and is identified in the electropheretogram of all seven HCC lines with various degrees of differentiation. In contrast, this atypical LD isoenzyme is not present in melanoma and all other tumor cells examined. In addition to LD,_, production, Hep G2 and Hep 3B, being classified as the most well-differentiated group, expressed only LDS isoform, which is characteristic of the hepatocyte-specific LD marker. As shown in Fig. 2, there was a tendency for higher levels of LD isoforms rich in H subunits in the less or poorly differentiated HCC cells. In contrast, the production of LD,_, appeared to be greater in the more differentiated HCC cells.
Melanoma
4. Discussion
Melanoma
In this study, we have made two novel findings. Firstly, we have shown that HCC cells at different stages of differentiation seem to show a distinct pattern of LD isoenzyme expression. In other words, there is a tendency of increasing the production of liver-specific M subunits at the expense of the cardiac-specific H subunit during the differentiation process. As an example, SK-Hep- 1,
Fig. 1. Electrophoretogram of LD isoenzymes in the cytosol fractions of cultured HCC cell lines having various degrees of differentiation. Normal human serum was used as a marker for proper assignment of the relative migrating positions of each isoenzyme. Two different melanoma cells were included for comparison. Arrow indicates the novel LD,_, isoenzyme expressed in all hepatoma cell lines examined.
196
T.Z. Liu et al. /Cancer
R
H subunit
Fig. 2. Relative composition differentiation.
w
of LD-M.
q LDH2-3/total
IS!! M subunit
Hep 38 Well
Lett. 87 (1994) 193-189
LD-H and LD,_,
PLC
J-5 differentiation
in the cytosol
being classified as the poorly-differentiated HCC cells, expressed an LD isoenzyme pattern resembling that of the normal human serum (5-isoform pattern). In contrast, it is interesting to point out that, even though the most poorly-differentiated HCC cells are capable of expressing more LD isoforms, yet their capability of synthesizing and secreting plasma proteins was completely turned off [6]. Our second novel finding reported here is a hepatoma-associated LD isoform which migrated electrophoretically to the relative position between LD, and LDs. There is an extensive literature about expression of isoenzymes, plasma proteins and antigens in human tumor tissue. These tumor-related syntheses of proteins can be categorized into two general types: in some cases, there appears to be a reversion in the pattern of gene expression characteristic of the fetal tissue. In other cases, there is an aberrant sythesis of proteins which appears to be due to the deregulation
fractions
Mahlavu of cultured
22T Poor HCC cell lines with various
degrees of
of genes which are not normally expressed [ 121. Our detection of this atypical hepatoma-associated LD isoenzyme, expressed in all of the hepatoma cells examined, seems to be consistent with the latter notion. Identification of the LD isoenzyme pattern, using the electrophoretic fluorescent scanning technique, depends on the oxidation of lactate substrate by the catalytic action of LD, which simultaneously reduces the non-fluorescent conenzyme, NAD, to its fluorescent counterpart, NADH. In order to prove the possibility that the observed atypical fluorescent band in the electrophoretogram was not an artifact, we initially stained the agarose gel film, after electrophoresis, with AMP buffer solution containing only NAD. With the omission of lactate substrate, we were able to show that not only the fluorescent bands associated with normal LD isoenzyme activities, but also the atypical one, had all totally disap-
T.Z. Liu et al. /Cancer
peared from the electrophoretogram (Data not shown). Thus we concluded that the atypical fluorescent band observed in the electrophoretogram was indeed due to the catalytic action of LD. Furinvestigators previously thermore, several observed the existence of macro LD isoenzyme complexes which usually consisted of an immunoglobulin bound to an LD isoenzyme [23-261. There are two such types of complexes, comprising LD bound to IgG and LD bound to IgA. In the presence of these complexes, the electrophoretic LD isoenzyme pattern will feature an abnormal number of isoenzyme bands, an altered electrophoretic mobility or a diffuse LD pattern. To determine whether the observed new variant of LD isoform is an immunoglobulin complex form, we separately added antisera of IgG, IgA, and IgE to the cell homogenates for 30 min prior to electrophoretic studies. Our results show that pretreatment with anti-immunoglobulin antibodies did not alter the fluorescent band of hepatomaassociated LD isoform (Data not shown). Thus, the possibility that this new form of LD isoenzyme is an immunoglobulin complex form can be ruled out. Further characterization of this hepatomaassociated LD isoform and evaluation of its potential clinical utility are currently under investigation. Acknowledgements
The authors are grateful to Ms. Jomei Tsai for her excellent work in typing this manuscript. This study was supported in part by grants from Chang Gung College of Medicine and Technology (CMRP406 and MRP365) and grants from the National Science Council of R.O.C. (NSC 820412-B 182-025 and NSC83-0412-B 182-033). References
111Knowles,
B.B., Howe, C.C. and Aden D.P. (1980) Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surfac antigen. Science, 209, 497-499. 121Kaighn, M.E. and Prince, A.M. (1971) Production of albumin and other serum protein by clonal cultures of normal human liver. Proc. Natl. Acad. Sci. USA, 68, 2396-2400.
Lett. 87 (1994) 193-189
19-l
131 Corlu, A., Kneip, B., Lhad, C., Leray, G., Baffet, G., Bourel, D. and Guguen-Guillouzo, C. (1991) A plasm membrane protein is involved in cell contact-mediated regulation of tissue-specific genes in adult hepatocytes. J. Cell. Biol., 115, 505-515. r41 Chang, C.M., Han, S.H. and Hu, C.P. (1984) Cellular antigenic properties of human hepatoma cell lines. In: Human Tumor Immunology, pp. 97-104. Editors: S.H. Han and C.S. Young. The Chinese Society of Immunology, Taipei. 151 Chang, CM., Hu, C.P., Tang, C.F., Chou, L.P., Chou, C.K. and Han, C.S. (1985) The differentiation properties of human hepatoma cell lines. In: Molecular Biology of Neoplasia, pp. 262-269. Editors: E.H. Chang J.K. Lin and P.C. Hung. Academia Sinica, Taipei. 161 Chang, C.M., Lin, Y .M., O-Lee, T.W., Chou, C.K., Lee, T.S., Liu, T.J., Peng, F.K., Chen, T.Y. and Hu, C.P. (1983) Induction of plasma protein secretion in a newly established human hepatoma cell line. Mol. Cell Biol., 3. 133-1137. K.G. and Mirkoritch, J. (1993) Gene (71 Xanthopoulos, regulation in rodent hepatocytes during development, differentiation and disease. Eur. J. Biochem., 216, 353-360. control 181 Lai, E. and Damell, J.E. (1991) Transcriptional in hepatocytes: a window on development, Trends Biochem. Sci., 16, 427-430. 191 DeSimone, V. and Cortese, R. (1991) Transcriptional regulation of liver-specific gene expression. Curr. Opin. Cell Biol., 3, 960-965. of 1101 Sladek, F.M. and Darnell, J.E. (1992) Mechanism liver-specific gene expression. Curr. Opin. Genet. Dev., 2, 256-259. S., Freiberg, A. and Amaral, L. (1973) [Ill Werthamer, Quantitation of lactate dehydrogenase isoenzyme patterns of the developing human fetus. Clin. Chim. Acta, 45, 5-11. Y.H. and Hopkinson, D.A. (1977) WI Edwards. Developmental change in the electrophoretic patterns of human enzymes and other proteins. In: Isoenzyme: Current Topics in Biological and Medical ‘Research, pp. 19-78. Editors: J.G. Scandalios and G.S. Whitt. Alan R Liss, New York. D31 Wilkinson. J.H. (1978) Plasma enzyme activities. In: Enzymes in Health and Disease, pp. 1-7. Proceedings Inaugural Scientific Meeting of the International Society for Clinical Enzymology, Karger, London. M. and Weber, F. 1141 Wood, D.C, Varela, V., Palmquist, (1973) Serum lactic dehydrogenase and isoenzyme changes in chronic cancer. J. Surg. Oncol., 5, 251-257. iI51 Rotenberg, Z., Weinberger, I. and Sugie, A. (1988) Total lactate dehydrogenase and its isoenzymes in serum of patients with non-mall-cell lung cancer. Clin. Chem., 34, 668-670. [I61 Balinsky, D.. Platz. C.E. and Lewis. J.W. (1983) Isoenzyme patterns of normal, benign and malignant human breast tissues. Cancer Res., 43, 5895-5901.
198
T.Z. Liu et al. /Cancer
[17] Strom, S. and Bendz, R. (1986) Creatine kinase and lactate dehydrogenase isoenzymes in stage D prostatic carcinoma. Clin. Chim. Acta, 159, 219-228. [I81 Liu, T.Z. (1985) Creatine kinase and lactate dehydrogenase isoenzyme patterns in some cultured colonic and pancreatic tumor cell lines [Abstract]. Clin. Chem., 31, 1004. [19] Aden, D.P., Fogel, A., Plotkin, S., Damjanov, I. and Knowles, B.B. (1976) Controlled synthesis of HBs Ag in a differentiated human liver carcinoma-derived cell line. Nature, 282, 615-616. [20] Alexander, J.J., Bey, E.M., Geddes, B.W. and Lecatsas, G. (1976) Establishment of a continuously growing cell line from primary carcinoma of the liver. S.A. Med. J., 50, 2121-2128. [21] Lin, Y., Hu, C.P., Chou, C.K., O-Lee, T.W., Wu, K.D., Chen, T.Y., Peng, F.K., Liu, T.J., Ko, L.L. and Chang, C.M. (1982) A new human hepatoma cell line: establish-
Lett. 87 (1994) 193-189
1221
(231
[24]
[25]
[26]
ment and characterization. Chiu, J. Microbial. Immunol., 15, 193-201. Fogh, J., Fogh, J.M. and Thomas, 0. (1977) One hundred and twenty-seven cultured human tumor cell lines producing tumor in nude mice. J. Natl. Cancer Inst., 59, 221-225. Biewenga, J. and Feltkamp, T.E.W. (1975) Lactate dehydrogenase (LDH)-IgG immunoglobulin complexes in human serum. Clin. Chim Acta, 64, 101-l 16. Acheampong-Mensah, D. (1980) Persistent increase of serum lactate dehydrogenase activity related to enzymeIgG(Lambda) immunoglobulin complex. Clin. Chem., 26, 789-790. Gorus, F., Aelbrecht W. and Van Camp, B. (1982) Circulating IgG-LD complex dissovable by addition of NAD+. Clin. Chem., 28, 236-239. Biewenga, J. and Feltkamp, T.E.W. (1975) LDH-IgA immunoglobulin complexes in human serum. Clin. Chim. Acta, 58, 239-249.
Letters
87 (1994) 193-198
Detection of a novel lactate dehydrogenase isozyme and an apparent differentiation-associated shift in isozyme profile in hepatoma cell lines T.Z. Liu”, P.Y. Chen”, D.T.Y.
Chiua, J.S. Wei”, K.S.S. Changb,
K.H. Lin*b
‘School of Medical Technology. Chang Gung College of Medicine and Technology. Kwei-Shari.. Taoyuan. Taiwan, ROC bGraduate Institute of Clinical Medicine, Chang Gung College of Medicine and Technology, Kwlei-Shan. Taoyuan, Taiwan, ROC
Received
26 August
1994; revision received
I I October
1994; accepted 12 October
1994
Abstract
A hitherto unreported lactate dehydrogenase (LD) isoenzyme, which migrates electrophoretically to the relative position between LD, and LD, has been identified in the electropheratogram in 7 of 7 (lOO’%) cultured hepatoma cell lines with various degrees of differentiation and is thus given the name LD,_,. LD2_3 seems to be specific for hepatoma cells because this atypical isoenzyme can not be detected in other tumor cell lines. In addition, the hepatoma cell lines also show a distinct pattern of LD isoenzyme and the isoenzyme pattern varies with the degree of differentiation. Hence, the expression pattern of LD isoenzyme phenotypes may provide a good marker for the investigation of human hepatoma cell differentiation. Keywork
Hepatoma
cells; Differentiation;
Atypical
LD isozyme; Electrophoretic
1. Introduction
The control of cell differentiation remains one of the most challenging problems in biology. However, an experimental model for studying the differentiation of hepatocytes is still lacking at present. The fact that a variety of plasma protein or plasma membrane protein markers of hepatocytes have been well characterized [l-5] has led Chang et al. [6] to propose an approach for study* Corresponding +Ol
author, Tel.: +Oll
886 3 3288263;
I 886 3 3280174.
0304-3835/94/$07.00 @ 1994 Elsevier Science Ireland SSDI 0304-3835(94)03601-E
Fax:
patterns
ing human hepatoma differentiation based upon the expression patterns of plasma proteins. However, this technique, though specific, is quite laborious and lengthy because of the necessity to analyze at least 15 different plasma proteins of the culture medium. Furthermore, a variety of hepatocyte-specific gene expressions have been shown to be closely regulated during differentiation. Regulation of the expression of gene encoding proteins that are secreted by the liver is predominantly controlled at the transcriptional level, and is mediated through the action of a set of hepatocyte-specific trans-acting factors [7- IO].
Ltd. All rights reserved
194
T.Z. Liu et al. /Cancer
These hepatocyte transcription factors may serve as the markers for differentiation. However, their assays can be quite cumbersome. Thus, it is desirable to find an alternative marker which is simple and rapid to perform in order to study differentiation of hepatocytes. Lactate dehydrogenase (LD; EC 1.1.1.27) is a glycolytic enzyme present in various tissues. It is well documented that LD is coded for by three genes, giving rise to M, H, and X subunits, while the X subunits are produced in all tissues, although in various amounts. Five isoenzymes of LD have been reported: homotetramers M, (LDJ and H4 (LD,), and heterotetramers M3H (LD& MzH, (LD,) and MH3 (LD3. It is generally accepted that cells undergoing transformation can have concurrent alteration of gene expression leading to the change of isoenzyme patterns. As an example, the isoenzyme patterns of LD are altered in the serum and in affected tissues of patients with neoplasma [13-l 71. Furthermore, Liu [ 181 compared the LD isoenzyme patterns of cultured colonic and pancreatic tumor cells and found that they had a preponderence of M subunits, ranging from 60 to 100% and had either LD4 or LD, as the predominant isoenzyme. In addition, the majority of tumor cells tested had been shown to be either completely devoid of or deficient in LDI. These studies indicate that the expression of LD isoenzyme patterns seems to be changeable and inducible depending on the state of proliferation. Thus far, to the best of our knowledge, the relationship between the degree of differentiation and production of LD isoenzyme patterns in cultured hepatoma cell lines has not yet been delineated. Herein we report the data of our investigation dealing with this interesting problem.
Lett. 87 (1994) 193-189
2.2. Preparation of cell homogenates Cells cultured in dishes (100 mm diameter) were washed two times with isotonic PBS (pH 7.4), scraped from the dish and resuspended in 1 ml of PBS. Twenty microliters of 0.1 M dithiothreitol solution was added to the cell suspension before sonication for 20 s in a cold ice bucket. The cell homogenates were centrifuged at 100 000 x g for 30 min and the supemate was centrifuged again and separated for use in the electrophoretic studies. 2.3. LD isoenzyme quantitation LD isoenzymes of the supematant fractions were separated electrophoretically using Corning agarose universal electrophoresis film. To ensure proper assignment of the relative migrating position of LD isoenzymes, the first lane of each electrophoresis film plate contained normal human serum as the marker. Sample size of application depended on the protein contents of supernatant fractions. Approximately equal amounts, normalized by the protein content of each supernatant, was applied to the assigned position of the electrophoresis film plate. Electrophoresis was carried out in a Corning universal barbital buffer containing 0.035% EDTA at pH 8.6 for 45 min. After electrophoresis, 1 ml of AMP lactate solution in 20% (w/v) sucrose (pH 9.0) was evenly dispensed onto the agarose surface along the edge of a serological pipette. After incubation for 15 min, the agarose gel film was oven-dried (60°C) and the LD isoenzyme profile was scanned using a fluorescence densitometer (Corning Model 740). The percentage of the M subunit was calculated as described previously by Balinsky et al. [16]: % of M =
2. Materials and methods 4(% LD.J + 3(%LD,)+ 2. I. CeN lines procurement and culture procedure Hepatoma cell lines were kindly provided by C.P. Hu (Cell Bank of Medical Research Unit of VA Hospital, Taiwan). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 0.1 mM non-essential amino acids. The cultures were maintained at 37°C in humidified air with 5% coz.
2(%LD,)
+ (%LD2)
4
3. Results Table 1 summarizes the sources and istics of seven lines of HCC cells. All of cells were previously classified into gories of differentiation depending on
characterthese HCC three catetheir mor-
T.Z. Liu et al. /Cancer Table I Origin and characteristics Cell line
of human
Sex
Group I (well-differentiated) Hep G2 M M Hep 3B
hepatoma
black
Group II (intermediate-differentiated) PLCIPRFIS African black M F Chinese J5 Group III (poorly-di@erentiated) African black Mahlavu M Chinese proteins HA22T M SK-Hep- 1 M Unknown ‘Lin,
K. H. et al. (unpublished
phologies and plasma protein Fig. 1 shows types of HCC entiation. For
References
Morphologically similar to typical hepatocytes and secrete approximately 15 plasma proteins
I,19 1,19
Morphologically similar to elongated hepatoBytes and secrete about 9 plasma proteins
20
Morphologically similar to fibroblast-like tocyte and secret 0 to 6 plasma proteins
20 21 22
Well
Hep 38 PLC I Differentiation
Mahlavu 221 1
SK-Hepl
hepa-
data)
sertJm control
J-5
195
Characteristics
capabilities of expressing various markers. the LD isoenzyme patterns of seven cells with various degrees of differcomparative purposes, we also in-
Hep G2
193-189
cell lines used in this study
Race
Caucasian American
Lett. 87 (1994)
Poor
eluded four colonic tumor cells (SW-620, SW-480, HCT-8, HCT-48), two pancreatic tumor cells (CAPAN-1, UCVA-1) and two melanoma tumor cells (H2169 T, H2058). As indicated in Fig. 1, an atypical LD isoenzyme (named LD,_, in this report) migrates electrophoretically to the relative position between LD2 and LD3 and is identified in the electropheretogram of all seven HCC lines with various degrees of differentiation. In contrast, this atypical LD isoenzyme is not present in melanoma and all other tumor cells examined. In addition to LD,_, production, Hep G2 and Hep 3B, being classified as the most well-differentiated group, expressed only LDS isoform, which is characteristic of the hepatocyte-specific LD marker. As shown in Fig. 2, there was a tendency for higher levels of LD isoforms rich in H subunits in the less or poorly differentiated HCC cells. In contrast, the production of LD,_, appeared to be greater in the more differentiated HCC cells.
Melanoma
4. Discussion
Melanoma
In this study, we have made two novel findings. Firstly, we have shown that HCC cells at different stages of differentiation seem to show a distinct pattern of LD isoenzyme expression. In other words, there is a tendency of increasing the production of liver-specific M subunits at the expense of the cardiac-specific H subunit during the differentiation process. As an example, SK-Hep- 1,
Fig. 1. Electrophoretogram of LD isoenzymes in the cytosol fractions of cultured HCC cell lines having various degrees of differentiation. Normal human serum was used as a marker for proper assignment of the relative migrating positions of each isoenzyme. Two different melanoma cells were included for comparison. Arrow indicates the novel LD,_, isoenzyme expressed in all hepatoma cell lines examined.
196
T.Z. Liu et al. /Cancer
R
H subunit
Fig. 2. Relative composition differentiation.
w
of LD-M.
q LDH2-3/total
IS!! M subunit
Hep 38 Well
Lett. 87 (1994) 193-189
LD-H and LD,_,
PLC
J-5 differentiation
in the cytosol
being classified as the poorly-differentiated HCC cells, expressed an LD isoenzyme pattern resembling that of the normal human serum (5-isoform pattern). In contrast, it is interesting to point out that, even though the most poorly-differentiated HCC cells are capable of expressing more LD isoforms, yet their capability of synthesizing and secreting plasma proteins was completely turned off [6]. Our second novel finding reported here is a hepatoma-associated LD isoform which migrated electrophoretically to the relative position between LD, and LDs. There is an extensive literature about expression of isoenzymes, plasma proteins and antigens in human tumor tissue. These tumor-related syntheses of proteins can be categorized into two general types: in some cases, there appears to be a reversion in the pattern of gene expression characteristic of the fetal tissue. In other cases, there is an aberrant sythesis of proteins which appears to be due to the deregulation
fractions
Mahlavu of cultured
22T Poor HCC cell lines with various
degrees of
of genes which are not normally expressed [ 121. Our detection of this atypical hepatoma-associated LD isoenzyme, expressed in all of the hepatoma cells examined, seems to be consistent with the latter notion. Identification of the LD isoenzyme pattern, using the electrophoretic fluorescent scanning technique, depends on the oxidation of lactate substrate by the catalytic action of LD, which simultaneously reduces the non-fluorescent conenzyme, NAD, to its fluorescent counterpart, NADH. In order to prove the possibility that the observed atypical fluorescent band in the electrophoretogram was not an artifact, we initially stained the agarose gel film, after electrophoresis, with AMP buffer solution containing only NAD. With the omission of lactate substrate, we were able to show that not only the fluorescent bands associated with normal LD isoenzyme activities, but also the atypical one, had all totally disap-
T.Z. Liu et al. /Cancer
peared from the electrophoretogram (Data not shown). Thus we concluded that the atypical fluorescent band observed in the electrophoretogram was indeed due to the catalytic action of LD. Furinvestigators previously thermore, several observed the existence of macro LD isoenzyme complexes which usually consisted of an immunoglobulin bound to an LD isoenzyme [23-261. There are two such types of complexes, comprising LD bound to IgG and LD bound to IgA. In the presence of these complexes, the electrophoretic LD isoenzyme pattern will feature an abnormal number of isoenzyme bands, an altered electrophoretic mobility or a diffuse LD pattern. To determine whether the observed new variant of LD isoform is an immunoglobulin complex form, we separately added antisera of IgG, IgA, and IgE to the cell homogenates for 30 min prior to electrophoretic studies. Our results show that pretreatment with anti-immunoglobulin antibodies did not alter the fluorescent band of hepatomaassociated LD isoform (Data not shown). Thus, the possibility that this new form of LD isoenzyme is an immunoglobulin complex form can be ruled out. Further characterization of this hepatomaassociated LD isoform and evaluation of its potential clinical utility are currently under investigation. Acknowledgements
The authors are grateful to Ms. Jomei Tsai for her excellent work in typing this manuscript. This study was supported in part by grants from Chang Gung College of Medicine and Technology (CMRP406 and MRP365) and grants from the National Science Council of R.O.C. (NSC 820412-B 182-025 and NSC83-0412-B 182-033). References
111Knowles,
B.B., Howe, C.C. and Aden D.P. (1980) Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surfac antigen. Science, 209, 497-499. 121Kaighn, M.E. and Prince, A.M. (1971) Production of albumin and other serum protein by clonal cultures of normal human liver. Proc. Natl. Acad. Sci. USA, 68, 2396-2400.
Lett. 87 (1994) 193-189
19-l
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