Expression of the Protein Product of the PCPH Proto-Oncogene in Human Tumor Cell LinesAuthor(s): Ana Rouzaut, Juan A. Recio, Vicente NotarioSource: Radiation Research, Vol. 155, No. 1, Part 2 (Jan., 2001), pp. 181-187Published by: Radiation Research SocietyStable URL: http://www.jstor.org/stable/3580400Accessed: 12/07/2010 10:43

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Expression of the Protein Product of the PCPH Proto-oncogene in Human Tumor Cell Lines

Ana Rouzaut, Juan A. Reciol and Vicente Notario2

Laboratory of Experimental Carcinogenesis, Department of Radiation Medicine, Georgetown University Medical Center, Washington, D.C. 20007

Rouzaut, A., Recio, J. A. and Notario, V. Expression of the Protein Product of the PCPH Proto-oncogene in Human Tu- mor Cell Lines. Radiat. Res. 155, 181-187 (2001).

Exposure of Syrian hamster embryo fibroblasts to chemical carcinogens resulted in the oncogenic activation of the PCPH proto-oncogene by induction of a single base-pair deletion that generated a truncated PCPH oncoprotein (mutated PCPH). Recently, we isolated and characterized the cDNA for the hu- man PCPH proto-oncogene and determined that in humans PCPH is a single-copy gene located in chromosome 14 (14q24.3). Pilot mRNA expression studies indicated that PCPH was expressed in the majority of normal organs tested, particularly in liver and kidney, but it appeared to be ex- pressed either at low levels or not at all in tumor cells or cell lines derived from the high-expressing tissues. We have gen- erated an antiserum against bacterial recombinant Syrian hamster PCPH. This antiserum recognizes both the normal and truncated, oncogenic Syrian hamster PCPH proteins and cross-reacts with the yeast, mouse, rat and human homologue proteins. Using this antibody, we have performed a study of PCPH expression in a larger sample of human neoplastic cell lines, including some derived from breast, nervous system, co- lon, lung and pancreas tumors. Results confirmed the frequent lack of PCPH expression in malignant cells and identified sev- eral immunoreactive forms of PCPH being differentially ex- pressed in cells of diverse tissue origins. ? 2001 by Radiation Research

Society

INTRODUCTION

In vitro transformation of Syrian hamster embryo cells to malignancy by exposure to physical and chemical car- cinogens (1) has been widely used as a model of experi- mental carcinogenesis that is relevant to studies of human cancer (2-4). Molecular analyses of the Syrian hamster cell system allowed us to establish that RAS activation occurs at low frequency in these cells (5), and to isolate PCPH

Present address: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.

2 Author to whom all correspondence should be addressed at Depart- ment of Radiation Medicine, Georgetown University Medical Center, Re- search Building, Room E215, 3970 Reservoir Road, NW, Washington, DC 20007.

(termed cph in the original reports), a novel oncogene ac- tivated upon 3-methylcholanthrene transformation using cosmid-rescue techniques (6). Further characterization stud- ies showed that PCPH is a single-copy gene localized on chromosome X in the hamster genome and that the Pcph oncogene acts synergistically with Hras in the transfor- mation of murine NIH 3T3 fibroblasts (6, 7). Furthermore, we showed that PCPH is conserved in eukaryotic cells, from yeast to humans, and that it is expressed in a broad

range of tissue types and developmental stages, from em- bryo cells to adult tissues in Syrian hamsters (7), suggest- ing that the normal function of the PCPH protein product may be essential for metabolic processes involved in the regulation of cell proliferation and survival.

More recently, we reported the isolation and sequencing of full-length cDNAs for the PCPH proto-oncogene and oncogene (mutated PCPH). Nucleotide sequence analysis confirmed that PCPH is indeed a novel gene (8). Compar- ison of the normal and mutated PCPH nucleotide sequences demonstrated that PCPH was activated by a single base- pair deletion within the coding region of the proto-onco- gene, causing a shift in the normal open reading frame, and the translation of the mutated protein to terminate 33 resi- dues downstream from the point-mutated base pair (8). Therefore, the mutated PCPH oncoprotein is a truncated form of the normal (246 compared to 469 amino acids) with an additional, rather hydrophobic C-terminal tail. Both nor- mal and mutated PCPH proteins showed partial homology to GTP/GDP exchange factors and nucleotide phosphatases. Biochemical analyses of in vitro translated normal and mu- tated PCPH proteins and functional analyses of cells trans- formed by mutated PCPH or transfected with PCPH ex- pression vectors indicated that (1) the normal and mutated products are ribonucleotide-binding proteins but do not cat- alyze nucleotide exchange on the HRAS protein, (2) steady- state levels of PCPH and particularly mutated PCPH mRNA are up-regulated in cells maintained in culture under serum deprivation, and (3) the mutated PCPH oncoprotein provides cells with enhanced stress-survival functions (8).

Although these results suggest that PCPH plays a role in mechanisms of cellular response to stress, the transforming function of the mutated PCPH oncogene and the normal function of the proto-oncogene remain to be elucidated. As

181

ROUZAUT, RECIO AND NOTARIO

A B C

Time post-transfection (h)

kDa U I I

57-

47-

30-

-.PCPH

- mt-PCPH

-GAPDH

FIG. 1. Characterization of the 367-10OW anti-PCPH antiserum. A: Detection of normal and mutated PCPH (mt-PCPH) (relative migration indicated by arrows on the far right) in total extracts of bacteria induced (I) to express the Syrian hamster proteins, but not in uninduced (U) cells. B: Detection of the endogenous S. cerevisiae (Sc) PCPH homologue (GDA1, -57-kDa polypeptide) and a mammalian PCPH (47 kDa) in a yeast transformant (Sc- PCPH) in which PCPH is expressed from a low copy-number vector. C: Detection of the transient expression (from 24 to 72 h) of the Syrian hamster normal and mutated PCPH after transfection into human 293T cells. Other endogenous PCPH-related polypeptides were also observed in untransfected (UT) cells. 3-Actin and GAPDH were used on stripped blots as loading controls.

a complementary approach to determine their function, we have isolated the mouse (9) and human (10) PCPH ho- mologues, established their pattern and level of mRNA ex- pression in adult tissues and during development of mouse embryos, and determined their localization on mouse and human chromosomes (9, 10). In this paper we report results from protein expression studies indicating that PCPH is dif- ferentially expressed in cells of a number of human tumor cell lines. These results suggest that alterations in the ex- pression of PCPH may be involved in the onset and/or development of a variety of human tumors.

MATERIALS AND METHODS

Cell Lines and Culture Conditions

Cells of 50 cell lines were obtained from either the American Type Culture Collection (Manassas, VA) or the Tissue Culture Core Facility of the Vincent T. Lombardi Cancer Center. Most cell lines were cultured in improved MEM supplemented with 10% fetal bovine serum and an- tibiotics (100 U/ml penicillin and 100 ,ig/ml streptomycin). Some of the cell lines were grown using MEM, Leibovitz L-15, McCoy's 5a, Dulbec- co's MEM or RPMI medium instead of improved MEM. All culture me- dium components were from Gibco (Life Technologies, Inc., Gaithers- burg, MD). Cells were incubated at 37?C in a humidified atmosphere of 95% air and 5% CO2.

Generation and Characterization of Anti-PCPH Antiserum

Escherichia coli BL21 (DE3) cells transformed with PCPH constructs were induced to express recombinant PCPH by the addition of 1 mM

isopropyl-D-thiogalactopyranoside (IPTG) for 1 h. The recombinant pro- tein was extracted from induced cells following the instructions in the His-Bind Buffer Kit (Novagen, Madison, WI), including the use of a 6 M urea extraction step because the PCPH protein fractionated with inclu- sion bodies. Recombinant PCPH was purified by one-step Ni2+ chelation chromatography on His-Bind Resin (Novagen), and the purified denatured protein was then refolded by a stepwise dialysis against a solution con- taining TBS (25 mM Tris-HC1, pH 7.4; 140 mM NaCI), 0.1% Triton X- 100, 5% glycerol, and progressively decreasing concentrations of urea, and without urea in the final step. After dialysis, the protein concentration was determined and the preparations were divided into aliquots and fro- zen at -80?C. An anti-PCPH polyclonal antiserum was generated by immunizing New Zealand rabbits by four sequential injections with a total of 1 mg of recombinant protein (performed by BioSynthesis, Inc., Lew- isville, TX). Blood samples collected at 6, 8 and 10 weeks after the first immunization were characterized for their ability to detect the normal and truncated PCPH proteins in total extracts from bacterial, yeast and mam- malian cells (Fig. 1). Specificity was checked by preincubating the anti- serum for 1 h at 4?C with purified, refolded bacterial recombinant PCPH to a final concentration of up to 100 ,xg of protein per ml of antiserum.

Western Immunodetection Analysis

When cultures reached 80% confluence, cells were scraped into harvest buffer (20 mM Tris-HCl, pH 7.5, 20 mM p-nitrophenyl phosphate, 1 mM EGTA, 50 mM sodium fluoride, 50 ,pM sodium orthovanadate, 5 mM benzamidine) and sonicated for 5 s. Total cell extracts (50 Ixg of protein) were subjected to SDS-PAGE on 4-15% gradient gels and blotted onto PVDF membranes by electrotransfer, and the membranes were probed with anti-PCPH antiserum as described (9, 10). Visualization of immu- noreactive polypeptides was accomplished by using a peroxidase-conju- gated secondary antibody and development with chemiluminescence (ECL, Amersham Pharmacia Biotech Inc., Piscataway, NJ). Blots were

182

'O.-C W'

4

P-Actin- 1

INVOLVEMENT OF PCPH IN HUMAN NEOPLASIA

stripped and reprobed with a polyclonal anti-p-actin antibody (Amer- sham) to control for equal sample loading. Steady-state levels of PCPH protein in normal epithelial cells were determined using a commercial blot (CellShot' Blot, from Clonetics, San Diego, CA) in which 75 ,ig of protein from total cell lysates of various human organs was added to each lane. The blot was processed as recommended by the manufacturer. The relative abundance of the immunoreactive polypeptides was deter- mined by densitometric analysis of the various blot exposures in a PDI Model DNA35 Scanner using PDI Discovery Series? software (Quantity One? version 2.0) for data processing.

RESULTS

Generation and Characterization of Anti-PCPH Antisera

Injection of purified bacterial recombinant PCPH into rabbits resulted in the generation of two antisera (nos. 366 and 367). Blood samples obtained weekly up to 10 weeks after immunization were compared for their ability to rec- ognize the bacterial recombinant Syrian hamster PCPH (51 kDa) and mutated PCPH (27 kDa) proteins in total extracts of IPTG-induced bacterial host cells (Fig. 1A, I lanes). Both 10-week antisera (366-10W and 367-10W) were able to

recognize specifically the induced proteins, but the 367- 10W antiserum was much more sensitive (worked at a 1: 5,000 dilution, Fig. 1A) than all other samples tested. Therefore, the 367-10W was used for further characteriza- tion studies. To test the ability of the 367-10W antiserum to detect PCPH proteins in more complex, eukaryotic back- grounds, we performed Western blot analyses on Saccha- romyces cerevisiae (Fig. 1B), immortal mouse and rat fi- broblasts (data not shown), and human 293T cells (Fig. 1C) before and after transfection with constructs (8, 11) de- signed for the expression of the products of the PCPH pro- to-oncogene (47 kDa) or the truncated mutated PCPH on- cogene (27 kDa). Results indicated that the 367-10W an- tiserum specifically recognized the endogenous yeast, mouse and human PCPH gene products as well as those encoded by the transfected PCPH constructs. These results conclusively demonstrated the ability of 367-10W to detect the PCPH proteins in total extracts of mammalian cells (Fig. 1).

PCPH Expression in Tumor Cell Lines

To determine the level and pattern of expression of PCPH in mammary tumor-derived cell lines, we used West- ern analysis of total extracts from a sample of 20 cell lines (Table 1). The 47-kDa PCPH polypeptide expected from the cDNA sequence of the wild-type human PCPH gene (10) was expressed either at low levels or not at all in 12 of the cell lines, with only 8 cell lines showing high or very high steady-state PCPH levels. Cell lines MDA-MB-361, MDA-MB-330 (Fig. 2A), ZR-75-1, MCF-7 (Fig. 2B), and MDA-MB-231 expressed particularly high levels of the 47 kDa PCPH polypeptide. Interestingly, we consistently ob- served the presence of other, sometimes major PCPH im- muno-related polypeptides of 90, 80, 70 and 27 kDa (Fig.

TABLE 1 PCPH Expression in Mammary Tumor Cell Lines

PCPH immuno-related polypeptides

Cell line 90 kDa 80 kDa 70 kDa 47 kDa 27 kDa

ZR-75-B - + ++ + ZR-75-1 - + - +++ + MDA-MB-157 - + ? + MCF-7 - +++ ++ + Hs-578T - + + + ++ BT-474 - - ++ ++ BT-479 - + + + + BT-20 - + - ++ - DU-4475 ? ++ + + + Hs-578BST + + + + +++ MDA-MB-361 - ++ ++ +++ + MDA-MB-330 ++ +++ + +++ ++ MDA-MB-134 + ++ + + 4

BT-483 + - ++ + ++ SK-BR-3 + + - - MDA-MB-231 - + +++ ++ MDA-MB-468 - - + + + MDA-MB-453 - - + - MDA-MB-436 - - + - + MDA-MB-415 - - - + +

Notes. (-) Undetectable; (+) detectable; (+) low; (+ +) high; (+ + +) very high.

high levels of most of these additional polypeptides (90, 80 and 27 kDa), whereas other cell lines accumulated high levels of only some of them. For example, Hs578T and BT483 cells expressed high levels of the 27-kDa polypep- tide, whereas MDA-MB-361 cells expressed high levels of the 70- and 80-kDa polypeptides (Fig. 1). The 27-kDa poly- peptide was detectable after prolonged exposures in all but two cell lines (BT20, Fig. 2B; and SK-BR-3, data not shown), whereas the distribution of the larger polypeptides (80 and 90 kDa) was still more restricted (Fig. 2).

Eighteen cell lines (Table 2) derived from the central or peripheral nervous system were analyzed by Western blot- ting with the 367-10W antiserum. Results showed that the 47-kDa PCPH polypeptide was either absent (13 cell lines) or barely detectable (4 cell lines) in the majority of the cases. Interestingly, the 27-kDa (13 cell lines) and 80-kDa (15 cell lines) polypeptides were expressed in most cell lines, whereas the 70-kDa polypeptide was present in only 7 cell lines. However, when present, the 70-kDa form was expressed at high levels in most cell lines.

Colon (six cell lines), lung (five cell lines), and pancreas (one cell line) cells were also included in this study. Over- all, results showed that PCPH is highly expressed in colon tumor cell lines (Table 3). All six colon cell lines showed very high levels of expression of the 47-kDa PCPH poly- peptide, and four of them also expressed high levels of the 27-kDa form. The larger forms were more poorly repre- sented but, when present, they were also expressed at high levels in most cases.

The overall level of expression of all PCPH immuno- related polypeptides was significantly lower in lung tumor-

183

2). Cell lines such as MDA-MB-330 (Fig. 2A) expressed

ROUZAUT, RECIO AND NOTARIO

A

(D CO C C') C')

0 2 2 .. " r II% I I I m 2 tr L << < c VW

, D DO q ? o I HO a 0 D m 0ED

PCPH

P-Actin .. .

B

PCPH

P-Actin

I - -

c r.a 0) c0

i O N N [ Z e 3 c o

" kDa

W80 <770

1-47

27

FIG. 2. PCPH expression in human tumor cell lines. Panels A and B correspond to Western blot analysis of total extracts from two representative sets of human breast carcinoma lines using the 367-1OW anti-PCPH antiserum. The

migration of the major PCPH immuno-related polypeptides is indicated, relative to a control consisting of a mixture of identical volumes of the 24-h extracts shown in Fig. 1C. P-Actin was used as the loading control.

kDa

--80 --70

- 47

184

INVOLVEMENT OF PCPH IN HUMAN NEOPLASIA

TABLE 2 PCPH Expression in Cell Lines Derived from the

Nervous System PCPH immuno-related polypeptides

Cell line 90 kDa 80 kDa 70 kDa 47 kDa 27 kDa

TC32 - - ++ - + T-98 - - - ++ SK-N-SH - + ++ - + SK-N-MC - + ++ - ++ RD - + - - ++ Hs-687 - + + + - DAOY - - - -

A-172 - ++ + - + H4 - + + U-87 - --

U-118 - ++ - - ++ U-138 - + - - ++ U-373 - ++ - + + IMR-32 - + + - - UW-2281 - + - - UW-2283 - + - + ++ UW-402 - + - + SY5Y - + ++ ++

Notes. (-) Undetectable; (?) detectable; (+) low; (++) high; (+ + +) very high.

derived cell lines than in cell lines of other tissue origin (Table 3). Cell lines such as NCI-82 did not seem to express any of the PCPH polypeptides, with the levels of the 47- kDa form approaching the lowest limit of detection of our technique, being detectable only after prolonged chemilu- minescence exposures. CALU cells represented the only case of a lung tumor cell line accumulating significant amounts of most major polypeptides. Interestingly, how- ever, two of the cell lines (SW900 and CALU, data not shown) expressed an additional PCPH immuno-related polypeptide of molecular mass of about 55 kDa, observed previously only in the MDA-MB-330 mammary tumor cell line (Fig. 2A).

The 47-kDa polypeptide was highly expressed in the PANC-1 pancreas carcinoma cell line, which showed levels of expression comparable to those detected in the mam- mary tumor cell lines MDA-MB-330, MDA-MB-361 (Fig. 2A), ZR-75-1, MCF-7 (Fig. 2B), and MDA-MB-231 or the colon tumor cell lines SL-174, SL-180 and HT-29 (Table 3). PANC-1 cells also accumulated high levels of the 70- kDa polypeptide and expressed minor additional PCPH im- muno-related polypeptides of about 35-40 and 55-60 kDa (data not shown).

PCPH Expression in Normal Epithelial Cells

Table 4 summarizes our findings on the expression of the various PCPH immuno-related polypeptides in the human tumor cell lines included in this study. The overall impres- sion is that, with the exception of the 90-kDa polypeptide, all other PCPH species are expressed in only about 50% of the cases. Because (1) these results support our previously

TABLE 3 PCPH Expression in Tumor Cell Lines from Colon,

Lung and Pancreas

PCPH immuno-related polypeptides

Cell line 90 kDa 80 kDa 70 kDa 47 kDa 27 kDa

Colon SL-174 + + ++ +++ +++ SL-180 + + ++ +++ +++ SW-480 - - - ++ SK-CO-1 - - ++ ++ HT-29 - +++ + +++ ++ H-620 - + + ++ +

Lung NCI-82 - - - + - NCI-520 - ++ + + + SW-900 - - + CALU - + + ++ H-1596 - + - +

Pancreas PANC-1 - + +++ ++ +

Notes. (-) Undetectable; (+) detectable; (+) low; (++) high; (+++) very high.

expressed notion (10) that the loss of PCPH in a high pro- portion of the tumor cells may play a role in neoplastic development, and (2) most tumors are epithelial in origin (12), we examined the expression of PCPH in extracts of primary human epithelial cells derived from various tissues. A commercial Western blot containing electrophoretically resolved extracts of epithelial cells from breast, two kidney regions, lung and prostate was probed with the 367-10W anti-PCPH antiserum. Results (Fig. 3) showed, to our sur- prise, that all epithelial cells tested expressed only the 27- kDa polypeptide. No evidence for the presence of the high- molecular-mass polypeptides was obtained by varying the conditions in our detection system. These results suggested that it may be the gain of the expression of the larger PCPH immuno-related polypeptides (47, 70 and 80 kDa) that re- ally is associated with the neoplastic phenotype rather than the lack of the 47-kDa polypeptide alone, as we proposed earlier (10).

TABLE 4 Summary of Data on PCPH Expression in Human

Tumor Cell Lines

PCPH immuno-related polypeptides Tumor origin 90 kDa 80 kDa 70 kDa 47 kDa 27 kDa

Breast 4/20 9/20 11/20 11/20 13/20 Nervous system 0/18 9/18 6/18 1/18 12/18 Colon 2/6 4/6 3/6 6/6 4/6 Lung 0/5 3/5 0/5 2/5 2/5 Pancreas 0/1 0/1 1/1 1/1 1/1 Overall 6/50 25/50 21/50 21/50 32/50 (%) 12.0 50.00 42.00 42.00 64.00

185

ROUZAUT, RECIO AND NOTARIO

PCPH - 27 kDa

1-Actin _.._ _ ... ? FIG. 3. Expression of PCPH in normal human epithelial cells. Western blot analysis with the 367-1OW anti-PCPH antiserum of a commercial blot

(Clonetics) containing total cell extracts from cultured epithelial cells isolated from the indicated organs. Only the 27-kDa PCPH form could be detected. P-Actin was used as the loading control.

DISCUSSION

We have prepared an anti-PCPH antiserum (367-10W) that specifically recognizes both normal and mutated pro- teins from Syrian hamster when expressed in bacteria, yeast or mammalian cells in addition to detecting the endogenous homologue proteins in these various species (Fig. 1), iden- tified on the basis of their molecular mass deduced from their published nucleotide sequences (10, 13). The com- petence and potency of our antiserum in the recognition of both normal and mutated PCPH in total cellular extracts were demonstrated by the ability of the antiserum to spe- cifically detect the transient expression of Syrian hamster normal and mutated PCPH after transfection into human 293T cells (Fig. 1C) at a dilution as high as 1:5,000. In fact, recent unpublished results have conclusively demon- strated that our 367-10W antiserum also works in immu- noprecipitation and immunohistochemistry.

Using this antiserum, we have completed a study of 50 human tumor cell lines to determine the level and pattern of PCPH expression in malignant cells. Overall, with regard to the 47-kDa PCPH polypeptide, the one with the molec- ular mass expected from the human PCPH cDNA nucleo- tide sequence, our results indicating lack of expression in 58% of the samples tested (Table 4) are in close agreement with previous data obtained in our laboratory (10) from the analysis of a smaller sample of human tumor cell lines of breast, liver, prostate and neuroectodermal origin that in- dicated that the 47-kDa polypeptide was not expressed in the majority (67.3%) of the cells tested. However, the in- clusion in this study of a larger sample has made it possible for us to detect other PCPH immuno-related polypeptides with the 367-10W antiserum in addition to the 47-kDa spe-

cies (Fig. 2). One of these polypeptides, the 90-kDa form, was detected only at low levels in a few cell lines, indi- cating that it is unlikely to be a relevant marker for neo- plastic development. Two other polypeptides (70 and 80 kDa) appear to be differentially expressed in certain types of tumor cell lines (Table 4). For instance, the 80-kDa poly- peptide was expressed more frequently in cell lines derived from the nervous system, and the 70-kDa form in breast tumor cell lines.

The structural relationship among the various PCPH im- muno-related polypeptides remains to be elucidated. We hy- pothesize that they may represent the translation products of alternatively spliced variants of the human PCPH mRNA. This hypothesis is based on our previous data (10) demonstrating that (a) PCPH is a single-copy gene in the human genome; (b) although the size of the most abundant PCPH transcript expressed in the majority of human tissues is about 4.5 kb in length, the full-length human PCPH cDNA was cloned from an mRNA of about 2 kb; (c) the cloned human PCPH cDNA encompassed partially spliced sequences from intron 4 of the PCPH gene; and (d) PCPH transcripts smaller than 4.5 kb were differentially expressed in various human tissues. Obviously, further experiments are required to understand the transcriptional and transla- tional regulation of the PCPH proto-oncogene.

An important observation in this study is the fact that only the 27-kDa PCPH polypeptide is expressed in normal epithelial cells (Fig. 3). Because of its similarity to the mo- lecular mass of the mutated PCPH oncoprotein (8), we had initially proposed (10) that its detection in human tumor cells could indicate the presence of truncated, putatively oncogenic forms of PCPH in the neoplastic cells. Such an

186

4ItsP

INVOLVEMENT OF PCPH IN HUMAN NEOPLASIA

interpretation has been re-evaluated in light of our present results. Although still larger samples of normal cells need to be examined, it seems that the disappearance of the 47- kDa form and/or the appearance of the 27-kDa form are not the main changes associated with neoplastic develop- ment. Indeed, the appearance of PCPH forms larger than 27 kDa represents the most common characteristic of all 50 cell lines studied. We did not identify any tumor cell line in this or previous studies that expressed only the 27- kDa PCPH form, in spite of it being the only PCPH poly- peptide detectable in normal epithelial cells.

There are reports in the literature, including some from our own laboratory (14, 15), indicating that alternative pat- terns of transcript and protein processing contribute to the onset and/or development of malignancy (16-18). Our re- sults raise the possibility that neoplastic development may be associated with alterations in the processing and matu- ration of PCPH transcripts and/or the post-translational pro- cessing of the PCPH proteins.

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service grant CA64472 from the National Cancer Institute. Densitometry was per- formed at the Lombardi Cancer Center's Macromolecular Synthesis and Sequencing Shared Resource. This and the Tissue Culture Resource of the Lombardi Center are supported in part by U.S. Public Health Service Grant P30-CA51008.

Received: November 29, 1999; accepted: February 15, 2000

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