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The Prostate 28:372-378 (I996)
There Are Multiple Forms of
Glyceraldehyde-3-Phosphate Dehydrogenase
in Prostate Cancer Cells and Normal
Prostate Tissue
Daniel E. Epner and Donald S. Coffey
Departments of Oncology ( D E E , D.S.C.), Urology (D.S.C.), and Biochemistry, Cellular,
and Molecular Biology (D.S.C.), The johns Hopkins University School of Medicine,
Baltimore, Maryland
We analyzed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in normal and malignant human prostate tissues, normal rat prostate, and Dunning R-3327rat prostate cancer cell lines. We detected multiple forms of GAPDH in Dunning
cell lines by two-dimensional protein electrophoresis and Western analysis. Five forms of
GAPDH that differed by isoelectric point were detected for each of the two metastatic
Dunning cell lines, while four or fewer forms were detected for Dunning cell lines with low
metastatic ability. We also detected multiple forms of GAPDH in normal and malignant
human prostate specimens by two dimensional protein electrophoresis and immunohistochemical analysis. GAPDH was undetectable in normal human prostate secretory epithelium by immunohistochemistry, but was abundant in nuclei of normal basal cells and stromal cells. In human prostate cancer specimens, there was a rough correlation between
cytoplasmic staining for GAPDH and tumor grade, but GAPDH staining was extremely
heterogeneous. GAPDH was abundant in nuclei of some high-grade human prostate tumors. Both of the human prostate cancer bone metastases analyzed with immunohistochemistry had markedly elevated cytoplasmic GAPDH expression. We conclude that multiple
forms of GAPDH may play diverse roles in normal prostate tissue and in prostate cancer.
0 1996 Wiley-Liss, Inc.
prostatic neoplasms, glyceraldehydephosphatedehydrogenase, immunohistochemistry, two-dimensional gel electrophoresis
GAPDH was initially identified several decades
ago as a key regulatory glycolytic enzyme [1,2]. In
recent years, there have been several studies that
show that GAPDH expression is increased in a variety of human tumors and cancer cell lines [3-81. We
previously showed that GAPDH RNA levels are increased in Dunning rat prostate tumors [9]. Since increased glycolysis is one of the hallmarks of cancer
[l], most authors have suggested that the role of
GAPDH in cancer cells is exclusively that of a glycolytic enzyme [3-81. However, GAPDH is now known
to have an astounding array of functions in both
transformed and nontransformed cells that are seem0 1996 Wiley-Liss, Inc.
ingly independent of its role in glycolysis. GAPDH is
a DNA repair enzyme [10,11], microtubule associated
protein [12-191, actin binding protein [20-231, protein
kinase [24], and substrate for epidermal growth factor
receptor kinase [25]. GAPDH is also thought to play a
role in detoxification of as-platinum and doxorubicin
in cancer cells [26]. We previously found a close cor-
Received for publication November 28, 1994; accepted June 16,
Address reprint requests to Dr. Daniel Epner, Baylor College of
Medicine, VA Medical Center, Medical Service (111H), 2002 Holcombe Blvd., Houston, TX 77030.
GAPDH and Prostate Cancer
relation between GAPDH RNA levels and metastatic
potential of Dunning rat prostate tumors, suggesting
that GAPDH may play a role in metastasis [9]. We
undertook this study to determine whether there are
multiple forms of GAPDH in normal and malignant
prostate cells that may play diverse roles in normal
prostate biology and prostate cancer progression.
Western Transfer
Semidry transfer of proteins to Hybond'"-ECL
nitrocellulose membranes (Amersham, Arlington
Heights, IL) with the Milliblot"-SDE transfer system
(Millipore) was done at 2 mA/cm2 for 30 min with
a discontinuous buffer system (anode buffer 1:300
mM Tris, 20% methanol, pH 10.4; anode buffer
2:25 mM Tris, 20% methanol, pH 10.4; cathode buffer:
25 mM Tris, 40 mM glycine, 20% methanol, pH 9.4).
Cell Culture
Dunning R-3327 rat prostate cancer cell lines,
which have been characterized previously [27-291,
were maintained in WMI, 10% fetal bovine serum,
dexamethasone 250 nM.
Immunoblotting was performed at room temperature. Nitrocellulose membranes were blocked for 1hr
in 10%nonfat dried milk/TBS-T (20 mM Tris, 137 mM
sodium chloride, 0.1% Tween-20, pH 7.6), washed
briefly with TBS-T alone, incubated for 1 hr with
monoclonal antibody to GAPDH (Biogenesis, Franklin, MA) diluted 1:1,000 in 5% nonfat dried milk/
TBS-T, washed several times with TBS-T, incubated
for 1 hr with horseradish peroxidase-labeled (Amersham) secondary antibody to mouse IgG, and
washed again several times with TBS-T. Filters were
then bathed in luminol and enhancer reagents (Amersham) according to the ECL'" Western blotting protocol for 1min and exposed to Kodak XAR-5 film for
10 sec-30 min.
Sample Preparation for Two-Dimensional Protein
Electrophoresis (2D PAGE) and Western Analysis
Adherent cells were washed twice with ice cold
phosphate buffered saline (PBS), incubated at 4°C for
45 min on a rocking platform in 1 ml of protein solubilization buffer (10 mM PIPES, 100 mM potassium
chloride, 1 mM EGTA, 2 mM magnesium chloride,
300 mM sucrose, 0.5% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 100 p,M leupeptin), scraped
from the dish, and placed in 1.5 ml conical polypropylene tubes. Normal rat ventral prostate was rinsed
in ice-cold PBS, snap frozen in liquid nitrogen,
ground in liquid nitrogen with a mortar and pestle,
and incubated for 45 min at 4°C in solubilization
buffer. The fresh frozen human tissue sample was
transferred directly to protein solubilization buffer
and incubated at 4°C for 45 min. All samples were
then centrifuged at 4"C, 10,000g for 10 min. The resultant supernatants, which contained >99% of total
cellular GAPDH protein (data not shown), were analyzed as described below.
High-resolution two-dimensional gel electrophoresis was performed with the Investigator 2-D gel system (MilligenBiosearch, Bedford, MA) as described
previously [30]. One hundred and twenty micrograms of Carbamylated CPK isoelectric point markers
(BDH Limited, Poole, UK) were included with each
sample. Isoelectric focusing was performed for 18,000
V-h using 1-mm x 16-cm tube gels. For the second
dimension, tube gels were then placed on 1-mm precast 10% Tris-acetate-SDS Duracryl polyacrylamide
slab gels (Millipore Co., Bedford, MA) and eledrophoresis was camed out at 12"C, 16,000 mW until the
bromophenol blue dye front was 17 cm from the top
of the gel (approximately 5 hr).
Patient Tissue Samples
A total of 13 paraffin-embedded patient samples
were analyzed for GAPDH expression by immunohistochemistry. Ten of the 13 were radical prostatectomy specimens removed from patients with clinical
stage B adenocarcinoma. Post-operative Gleason
scores for these 10 prostatectomy specimens ranged
from 5-9. One sample was obtained from a 31-yearold man who underwent cystoprostatectomy for
bladder disease and who was incidentally noted to
have a small focus of adenocarcinoma of the prostate
with Gleason score 1 2 = 3. The remaining two
specimens were bone metastases resected from patients with stage D adenocarcinoma of the prostate.
The fresh frozen radical prostatectomy specimen
tested by 2D PAGE was from a patient with stage B
adenocarcinoma, Gleason grade 7. This specimen
was cut into serial 6 Fm sections and examined by
hematoxylin and eosin staining at approximately 100
p,m intervals to identdy areas of nearly pure cancer or
normal tissue. Cut sections were stored at -80°C until the time of analysis, when they were solubilized as
described above.
Immunohistochemistry was performed with the
automated Bio Tek Techmate 1,000 system (Santa Bar-
Epner and Coffey
bara, CA). Six micron sections of paraffin-embedded
specimens were mounted on Chem Mate Capillary
Gap Plus microscope slides and microwave treated.
Sections were deparaffinized by sequential treatment with xylene, absolute ethanol, 95% ethanol,
and 80% ethanol; incubated with hydrogen peroxide
to eliminate endogenous peroxidases and decrease
background staining; incubated sequentially with
monoclonal antibody to GAPDH diluted 1:2,000 (Biogenesis), followed by biotinylated secondary antibody, followed by avidin and peroxidase-complexed
tertiary antibody; and exposed to diaminobenzene, a
chromagen which yields a brown color.
There Are Multiple Forms of GAPDH Detectable
in Dunning Prostate Cancer Cell Lines by
Two-Dimensional Protein Electrophoresis (2D
PAGE) and Western Analysis
Results of 2D PAGE and Western analysis are
shown in Figure 1. There were five forms of GAPDH
detected in the two highly metastatic cell lines tested
(MatLyLu and MatLu), while no more than four
forms of GAPDH were detected in cell lines with low
metastatic ability. Only one major form of GAPDH
was detected in the G cell line, which is the most
indolent and only androgen-sensitive Dunning cell
line. GAPDH protein, which is ubiquitous, could not
be detected in normal rat ventral prostate tissue under the conditions of the experiment. The absence of
detectable GAPDH protein in samples from normal
rat prostate illustrates the degree to which GAPDH
expression is increased in cancer cells. Isoelectric
standards were included in each sample to allow accurate assignment of PI values.
The results shown in Figure 1are not quantitative,
but instead show the relative abundance of various
forms of GAPDH in the different cell lines. Twice as
much total protein was loaded for G cells and normal
prostate as for the other cell lines. Results of quantitative, one-dimensional Western analysis of four
Dunning cell lines are shown in Figure 2. It is apparent from Figure 2 that GAPDH is much less abundant
in nonmetastatic G cells than in other Dunning cell
lines, and that total GAPDH protein levels do not
correlate with metastatic potential.
Human Prostate Cancer Tiswe I s Distinguishable
From Normal Prostate Tissue by 2D PAGE and
Western Analysis for GAPDH
As shown in Figure 3, there were at least two distinct forms of GAPDH with PI of approximately 7.05
and 7.1 detected in normal human prostate tissue. In
lsoelectric focusing
37 kD
37 kD
High Metastatic Potential
37 kD 37 kD
37 kD
37 kD
Fig. I. 2D PAGE and Western analysis of Dunning cell lines for
GAPDH protein. The patterns were aligned based on the location
of pl markers which were included in each sample.
37 kD -
Optical Density
Fig. 2. I D PAGE and Western analysis of GAPDH levels in Dunning cell lines. Forty micrograms of total protein were loaded per
lane. Corresponding optical density measurements are indicated
directly beneath each band. Cell lines with low metastatic potential: G, ATI, and ATZ; cell line with high metastatic potential:
Mat-LyLu (MLL).
addition, there was a broad band of signal in the PI
range of 6.75-6.9 which may either represent multiple forms of GAPDH of low abundance or one form
which does not focus sharply. In contrast, the cancer
specimen from the same patient did not contain the
GAPDH and Prostate Cancer
lsoelectric focusing
PI 7.0
TABLE 1. Summary of ImmunohistochemicalAnalysis of
GAPDH Expression in Human Prostate Tissue*
Normal tissue
Basal cells
Secretory cells
Stromal cells
Low grade
High grade
Metastasis (bone)
37 kD -
37 kD -
*-, none;
Fig. 3. 2D PAGE and Western analysis of human prostate specimens for GAPDH protein.
PI 7.1 form, but instead had a prominent signal at PI
7-7.05 that appeared to represent two forms of
GAPDH with slightly different isoeledric points. The
broad band in the PI range of 6.75-6.9 was also
present in the cancer specimen.
lmmunohistochemical Analysis of Human Prostate
Tissue Reveals Multiple Forms of GAPDH With
Nuclear and Cytoplasmic Localization
Results of immunohistochemical analysis of
GAPDH expression in human prostate tissue are
summarized in Table I. GAPDH was abundant in all
basal cell nuclei and in many stromal cell nuclei of all
normal tissues studied (Fig. 4a). In contrast, GAPDH
was not detectable in normal secretory epithelium
(Fig. 4a).
While GAPDH staining of normal prostate epithelium was uniform for all patients, GAPDH staining of
prostate cancer specimens was extremely heterogeneous. Most low-grade cancers stained faintly for
GAPDH (Fig. 4b), but some stained with moderate
intensity. All high-grade tumors had moderate to intense nuclear or cytoplasmic staining for GAPDH
(Figs. 4c and d, respectively), and some tumors had
both nuclear and cytoplasmic staining. While there
was a rough correlation between staining intensity
and histologic grade, there was no consistent relationship between GAPDH staining and long-term
clinical outcome (data not shown). Both metastatic
lesions had very intense cytoplasmic GAPDH staining (Fig. 4e).
We found that five forms of GAPDH with different
isoelectric points can be detected in metastatic Dun-
+ , little; + +, moderate; + + +, much.
ning rat prostate cancer cell lines, while one to four
forms can be detected in nonmetastaticcell lines (Fig.
1).We also detected at least three forms of GAPDH
by 2D PAGE of human prostate tissues (Fig. 3). All of
the forms of GAPDH detected in these studies represent 37 kD monomers. Since GAPDH is a tetramer
in vivo, there may be many different GAPDH isoenzymes present in prostate cancer cells [31,32], each of
which could conceivably have a unique function.
There may also be GAPDH isoenzymes that are specific for prostate cancer cells.
Further experiments will be required to determine
whether the various forms of GAPDH in prostate
cancer cells result from transcriptional activation of
genes that are normally silent, posttranslational
modification of a single gene product, or both. The
different forms of GAPDH may represent differently
phosphorylated proteins with identical amino acid
sequence, since GAPDH is known to undergo autophosphorylation [24] and to be phosphorylated by
epidermal-growth-factor-receptor kinase [25] and
Ca’+/calmodulin-dependent protein kinase I1 [33].
Phosphorylation of GAPDH may lead to nuclear localization, as is the case for protein kinase C and
other proteins [34,35].
The fact that GAPDH is abundant in nuclei of normal prostate basal cells suggests that it may play a
role in DNA repair in prostate epithelium, as it does
in other tissues. High levels of cytoplasmic GAPDH
in prostate cancer cells are probably at least partially
a reflection of elevated glycolysis, one of the hallmarks of cancer. However, cytoplasmic GAPDH may
have nonglycolytic roles in prostate cancer cells, since
it is known to have an astounding number of diverse
functions in other tissues. Future work will aim to
clarify the roles that GAPDH has in prostate tissue
and to determine whether there are cancer-specific
GAPDH isoenzymes.
Epner and Coffer
Fig. 4. lmmunohistochemical analysis of GAPDH expression in human prostate specimens. a:
Normal gland, x 100; b low-grade adenucarcinoma. x 64 (bar = 40 km); c: nuclear staining in
high-grade adenocarcinoma. x 64; d: cytoplasmic staining in high-grade adenocarcinoma X 64; e:
bone metastasis, x 100.
1. There are multiple forms of GAPDH in normal
and malignant human prostate tissues and in rat
prostate cancer cell lines.
2. Since GAF'DH is known to have many diverse
functions, it may have multiple roles in prostate can-
cer that are independent of its established role in glycolysis.
Dr.Epner is the recipient of an American Cancer
Society Physician's Research Training Award (PRTA-
GAPDH and Prostate Cancer
14). This work was also supported by National Cancer Institute Grant CA15416; National Institute of
Arthritis, Metabolism, and Digestive and Kidney Diseases Grants DK07552 and DK19300 (Department of
Health and Human Services); and a SPORE grant for
prostate cancer. The authors thank Dr.Jonathan Epstein for assistance in interpretation of immunohistochemical studies and Dr. Steven Bova for microdissecting the fresh frozen patient specimen analyzed by
2D PAGE and Western analysis.
1. Warburg OH: “The Metabolism of Tumors.” [Translated from the German edition by F. Dickens.] London:
Constable, 1930.
2. Stryer, L: Biochemistry. New York: W.H. Freeman and
Company, 1995.
3. Tokunaga K, Nakamura Y, Sakata K, Fujimori K,
Ohkubo M, Sawada K, Sakiyama S: Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase
gene in human lung cancers. Cancer Res 475616-1519,
4. Schek N, Hall BL, Finn OJ: Increased glyceraldehyde3-phosphate dehydrogenase gene expression in human
pancreatic adenocarcinoma. Cancer Res 48:6354-6359,
5. Persons DA, Schek N, Hall BL, Finn OJ: Increased expression of glycolysis-associated genes in oncogenetransformed and growth-accelerated states. Mol Carcinog 288-94, 1989.
6. Perfetti V, Manenti G, Dragani TA: Expression of
housekeeping genes in Hodgkin’s disease lymph
nodes. Leukemia 5:lllO-1112, 1991.
7. Desprez PY, Poujol D, Saez S: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1. 12.) gene
expression in two malignant human mammary epithelial cell lines: BT-20 and MCF-7. Regulation of gene expression by 1,25-dihydroxyvitaminD, (1,25-(OH),D,).
Cancer Lett 64:219-224, 1992.
8. Ohkubo M, Nakamura Y, Tokunaga K, Sakiyam S: Similarity between glyceraldehyde-$phosphate dehydrogenase and a 37,000-dalton protein which is abundantly expressed in human lung cancers. Jpn J Cancer
Res 77:554-559, 1986.
9. Epner DE, Partin AW, Schalken JA, Isaacs JT, Coffey
DS: Association of glyceraldehyde-3-phosphate dehydrogenase expression with cell motility and metastatic
potential of rat prostatic adenocarcinoma. Cancer Res
53:1995-1997, 1993.
10. Meyer-Siegler K, Mauro DJ, Seal G, Wurzer J, DeRiel
JK, Sirover MA: A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase. Proc Natl Acad Sci USA 88:
11. Ronai Z: Glycolytic enzymes as DNA binding proteins.
Int J Biochem 25:1073-1076, 1993.
12. Muronetz VI, Wang Z-X,Keith TJ, h u l l HR, Srivastava DK: Binding constants and stoichiometries of glyceraldehyde-3-phosphate dehyrogenase-tubulin complexes. Arch Biochem Biophys 313:253-260, 1994.
13. Somers M, EngelborghsY, Baert J: Analysis of the binding of glyceraldehyde-3-phosphate dehydrogenase to
microtubules, the mechanism of bundle formation and
the linkage effect. Eur J Biochem 193:437-444, 1990.
14. Aithal NH, Walsh-Reitz MM, Kartha S, Janulis MP,
Martin TE, Toback FG: Glyceraldehyde-3-phosphate
dehydrogenase modifier protein is associated with microtubules in kidney epithelial cells. Am J Physiol266:
F612-619, 1994.
15. Dumeu C, Bernier-Valentin F, Rousset B: Microtubules
bind glyceraldehyde 3-phosphate dehydrogenase and
modulate its enzyme activity and quaternary structure.
Arch Biochem Biophys 252:32-40, 1987.
16. Balaban N, Goldman R The association of glycosomal
enzymes and microtubules: a physiological phenomenon or an experimental artifact? Exp Cell Res 191:219226, 1990.
17. Walsh JL, Keith TJ, Knull HR: Glycolytic enzyme interactions with tubulin and microtubules. Biochim Biophys Acta 99954-70, 1989.
18. Launay JF, Jellali A, Vanier MT: Glyceraldehyde-3phosphate dehydrogenase is a microtubule binding
protein in a human colon tumor cell line. Biochim Biophys Acta 996:103-109, 1989.
19. Huitorel P, Pantaloni D: Bundling of microtubules by
glyceraldehyde-3-phosphate dehydrogenase and its
modulation by ATP. Eur J Biochem 150:265-269, 1985.
20. Masters CJ, Reid S, Don M: Glycolysis-new concepts
in an old pathway. Mol Cell Biochem 76:3-14, 1987.
21. Beitner R Regulation of carbohydrate metabolism.
Boca Raton, FL: CRC Press, Inc., 1985.
22. K n d HR, Walsh JL: Association of glycolytic enzymes
with the cytoskeleton. Curr Top Cell Regul 33:15-30,
23. Masters C Interactions between glycolytic enzymes
and components of the cytomatrix. J Cell Biol99:222s
225.5, 1984.
24. Kawamoto RM, Caswell AH: Autophosphorylation of
glyceraldehydephosphate dehydrogenase and phosphorylation of protein from skeletal muscle microsomes. Biochemistry 25656-661, 1986.
25. Reiss N, Kanety H, Schlessinger J: Five enzymes of the
glycolytic pathway serve as substrates for purified epidermal-growth-factor receptor kinase. Biochem J 239:
691-697, 1986.
26. Hao XY, Bergh J, Brodin 0, Hellman U, Mannervik B:
Acquired resistance to cisplatin and doxorubicin in a
small cell lung cancer cell line is correlated to elevated
expression of glutathione-linked detoxification enzymes. Carcinogenesis 15:1167-1173, 1994.
27. Isaacs JT, Isaacs WB, Feitz WFJ, Scheres J: Establishment and characterization of seven Dunning rat prostatic cancer cell lines and their use in developing methods for predicting metastatic abilities of prostatic
cancer. Prostate 9:261-281, 1986.
28. Isaacs JT, Heston WDW, Weissman RM, Coffey DS:
Animal models of the hormone-sensitive and -insensitive prostatic adenocarcinomas, Dunning R-3327-H,
R-3327-HI, and R-3327-AT. Cancer Res 384353-4359,
29. Isaacs JT, Wake N, Coffey DS, Sandberg AA: Genetic
instability coupled to clonal selection as a mechanism
for tumor progression in the Dunning R-3327 rat prostatic adenocarcinoma system. Cancer Res 42:23532361, 1982.
30. Patton WF, Pluskal MG, Skea WM, Buecker JL, Lopez
MF, Zimmerman R, Belanger LM, Hatch PD: Develop-
Epner and Coffey
ment of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility
and polypeptide resolution. Bio Techniques 8518-527,
31. Edwards YH, Clark P, Hams H: Isozymes of glyceraldehyde-%phosphate dehydrogenase in man and other
animals. Ann Hum Genet 40:67-77, 1976.
32. Weinhouse S, Shatton JB, Criss WE, Farina FA, Morris
Hp: Isozymes in relation to differentiation in transplantable rat hepatomas. In Weinhouse S, Ono T (eds):
”Isozymes and Enzyme Regulation in Cancer.” Tokyo:
University of Tokyo Press, 1972, pp 1-17.
33. Ashmarina LI, Louzenko SE, Severin SE Jr, Muronetz
VI, Nagradova NK Phosphorylation of D-glyceraldehyde-3-phosphate dehydrogenase by Ca2+/calmodulin-dependent protein kinase II. FEBS Lett 231:413-416,
34. Dingwall C, Laskey R The nuclear membrane. Science
258:942-947, 1992.
35. Silver PA: How proteins enter the nucleus. Cell 64:489497, 1991.
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