EXPERIMENTAL CELL RESEARCH 240, 236–243 (1998)ARTICLE NO. EX983940

Elevated Expression of the cdc25A Protein Phosphatasein Colon Cancer

Dora Dixon,* Terence Moyana,† and Martin J. King*,†,1

*Saskatoon Cancer Centre, 20 Campus Drive, and †Department of Pathology, University of Saskatchewan,Saskatoon, Saskatchewan, Canada S7N 4H4

transformation [4]. In addition to this the cell cycleThe nuclear protein phosphatase cdc25A has been PTPs cdc25A and cdc25B have been postulated to be

postulated to be a protooncogene. The total nuclear protooncoproteins. When expressed in conjunction withphosphotyrosyl protein phosphatase (PTP) activity H-ras or the absence of the tumor suppressor RB1 theand the expression of cdc25A were compared in nor- overexpression of cdc25A and cdc25B resulted in cellu-mal and cancerous colon epithelial tissue. Nuclei de- lar transformation of mouse fibroblasts [5]. cdc25A reg-rived from normal mucosal epithelium and tumors ulates the progression of the cell cycle from G1 to Swere analyzed for phosphotyrosyl protein phospha- phase, consequently cdc25A plays a central role in thetase activity using the malachite green assay and a decision as to whether a cell divides or not.synthetic phosphotyrosyl peptide based on the se- The importance of PTP activity in signal transduc-quence of cdc2, a known cdc25A phosphotyrosyl pro- tion and cancer was initially shown by incubatingtein substrate. Tumorigenesis resulted in elevated nu- NRK-1 cells [6] or quiescent mouse lens cells [7] withclear PTP activity (343.0 { 37.0% of normal epithelial

sodium orthovanadate (a PTP inhibitor). In each casePTP activity) in 52% (29 of 56) of colon tumors. In allthis led to an increase in the phosphotyrosyl proteincases elevated nuclear PTP activity correlated with ancontent and the induction of transformation of theseincrease in the expression of cdc25A. The changes incells. However, there is no consensus of action of PTPsPTP activity observed were not due to any increase inin cancer; i.e., PTPs have been shown to be elevated inthe rate of growth of the colonic mucosa as no corre-certain tumors yet act as growth inhibitors, or antitu-sponding changes occurred with PTP activity undermor agents, in other cells and cancers. Consequentlyconditions of rapid mucosal growth. q 1998 Academic Press

the picture is far from clear. For example, as mentionedKey Words: cdc25A; phosphotyrosyl protein phospha-above both cdc25A and cdc25B have been postulatedtase; colon cancer; nuclei; mitotic index.to be protooncoproteins, with cdc25B being overex-pressed in 30% of human breast cancers [5]. Also theoverexpression of PTP 1B appears to be a common phe-INTRODUCTIONnotype (72–80% of tumors investigated) in human

Cellular protein phosphotyrosyl levels are controlled breast [8, 9] and ovarian cancer [10], indicating thatby a balance between tyrosine kinase and phosphotyro- elevation of PTP 1B may represent a common patho-syl protein phosphatase (PTP) activities. Increases in genical pathway. Abnormal expression of PTP 1B cor-the protein phosphotyrosyl concentration in a cell are related with a poor prognosis in these cancers. In fact,frequently associated with cellular growth [1, 2]. How- aberrant expression of PTP 1B had a stronger correla-ever, increases in PTP activity may not always be con- tion with adverse clinical outcome than the conven-sistent with growth inhibition [3, 4]. For example, stim- tional oncogene markers, i.e., p185c0erb B2 [8, 10]. Ele-ulation of CD45 and PTPa is known to stimulate the vated PTP expression (PTP 1B and LAR) was also ob-activity of certain members of the src family of tyrosine served in three human breast cell lines overexpressingkinases by the removal of the inhibitory phosphotyrosyl the activated neu oncogene and in a rat mammary car-residue (for c-src; tyrosine 527 in chickens and tyrosine cinoma expressing high levels of neu [11]. In each case530 in humans), resulting in an overall increase in the PTP expression correlated with the expression of neuphosphotyrosyl concentration in the cell and cellular and tumorigenicity [11]. SAP-1 (stomach cancer-associ-

ated PTP; a receptor PTP structurally similar toHPTPb) was observed to be highly expressed in five1 To whom correspondence and reprint requests should be ad-colon cancer cell lines and two pancreas cancer celldressed at: Saskatoon Cancer Centre, 20 Campus Drive, Saskatoon,

Saskatchewan, Canada S7N 4H4. Fax: (306) 655-2910. lines but not in normal colon or pancreas, indicating

2360014-4827/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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237cdc25A IN COLON CANCER

fed state and the large bowel was excised and placed in ice-cold saline.that SAP-1 may be involved in the carcinogenesis ofThe colon was measured and divided into ascending and descendingthese cells [12]. It has recently been demonstrated thatcolon based on physiology (the ascending colon possesses herringbonethe balance between tyrosine kinase/PTP expression markings); the distal 2 cm of the colon was termed rectum and was

levels as well as subcellular localization is an im- processed with the descending colon. The mucosa was scraped fromthe smooth muscle and homogenized in 5 vol buffer A [25 mM Tris/portant factor in the ability of a specific PTP to inhibitHCl, pH 7.4, containing 5 mM EDTA, 1 mM EGTA, 1 mM DTT, 1cell transformation [13]. The overexpression of the nu-mM benzamidine, 20 mg/ml soybean trypsin inhibitor, 1 mg/ml leupep-clear PTP, PRL-1, in NIH 3T3 cells resulted in cellulartin, 10 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride]. The

transformation, as demonstrated by an increased cell homogenate was centrifuged at 500g for 20 min. The resulting super-growth, increased cell density, and the formation of natant was used to prepare the postnuclear subcellular fractions (see

below) while the pellet was used to prepare whole nuclei based oncolonies in soft agar [3], indicating that this PTP posi-the method of Chauveau et al. [18] in buffer A containing 1.8 Mtively regulated cellular growth. PRL-1 is also elevatedsucrose. Whole nuclei were stored at 0207C in Tris buffer containingat all times in actively dividing cells, being expressed 40% glycerol. Under these conditions they remain whole and viable.

at high levels during liver development and regenera- Whole nuclei possessed a low endogenous phosphate background andtion as well as in several tumor cell lines [3]. consequently did not require dialyzing prior to PTP activity assays.

Protein was determined by the dye-binding assay with BSA as proteinConversely, the overexpression of PTP 1B on NIHstandard as described by King and Sharma [19].3T3 cells transformed with neu resulted in an inhibi-

Phosphotyrosyl protein phosphatase assay. Colon PTPs were as-tion of cell transformation [14] and the overexpressionsayed using synthetic phosphotyrosyl peptides. Phosphotyrosyl pep-of a bovine PTP in normal and v-erb B2 transformed tide dephosphorylation was performed as described [20]. Briefly,

NIH/3T3 cells resulted in an inhibition of proliferation phosphopeptide (100–200 mM) was incubated with cellular extractsof both the cell lines [15]. It is of interest to note that (0.08–0.8 mg/ml) for 2–60 min in the presence or absence of various

effectors, as indicated, in a final volume of 25 ml. The reactions weretwo antitumor agents, gallium nitrate and suramin,initiated by the addition of cell extract and terminated by the addi-are able to potently inhibit certain PTP activities fromtion of 100 ml malachite green solution. The color was allowed toleukemia, prostate, and colon cancer cell lines [16, 17]. develop for at least 15 min before being read at 655 nm on a Bio-

The observations to date indicate that PTPs may be Rad microplate reader (Model 3550). The reaction color was stableelevated in certain cancerous cells, possibly opposing for 12 h. Reactions were restricted to õ30% dephosphorylation, in

the linear range of dephosphorylation. All PTP reactions were per-or acting in concert with tyrosine kinases, yet act asformed in duplicate for subcellular extracts or triplicate for wholegrowth inhibitors in others. Whether a specific PTPnuclei. All reagents used were initially tested with malachite greenacts as a protooncogene or an antioncogene may be to ensure low backgrounds and compatibility with the phosphatase

dependent on the cell type and the expression, or loss, reaction. Dephosphorylation units are described as either nmol Pireleased/min/mg or percentage of control.of other regulatory proteins, i.e., H-ras. The loss of a

specific PTP, a potential mechanism for tumorigenesis, Induction of colon cancer. Colon cancer was induced in Sprague–Dawley rats (150–200 g) by 8 weekly i.p. injections of azoxymethanewould have a similar effect as the gaining, or constitu-(AOM; 10 mg/kg body wt) in phosphate-buffered saline. Control ratstively switching on, of a tyrosine kinase. To date muchwere injected with saline. Colon tumors developed after 3 monthswork has focused on the roles of the tyrosine kinases; from the completion of the AOM injections (the sacrifice of the rats

this is in sharp contrast to the paucity of studies con- occurred 84 days after the last AOM injections). Rats were sacrificedin a fed state and the colons removed as described above. The colonscerning the roles of the PTPs. The objective of this pres-were opened and the size and position of each tumor recorded. Adeno-ent study is to investigate the role of nuclear PTP activ-mas and adenocarcinomas were dissected from the colon. Normality and cdc25A, in particular, in colon epithelial mucosaappearing colon from these rats was handled similar to the controlby comparing activity and level of expression of PTPs epithelial mucosa (described above). Tumors were divided into two

in normal and cancerous colon epithelial tissue. segments by a vertical cut. Half of the tumor was fixed in 4% formal-dehyde for 18–24 h for histological analysis while the remainingsegment was used for the analysis of PTP activity. Tumors analyzedEXPERIMENTAL PROCEDURES for PTP activity were homogenized and the cellular fractions pre-pared were assayed immediately or were stored at 0207C (whole

Materials. Sprague–Dawley rats were obtained from Charles nuclei) or 0807C (cell extracts) until use. Fixed tumor segments wereRiver, Inc. The phosphotyrosyl peptides were obtained from Up- embedded in paraffin wax, sectioned (5 mm thickness), mounted onstate Biotechnology [src416, RRLIEDAEY(p)AARG; src527, TST- microscope slides, and stained with hematoxylin and eosin. The le-EPQY(p)GENL] or Genetic Research [cdc215, MTGDTY(p)TAHAG; sions were graded into three categories: normal mucosa; tumor, noabl393, EKIGTY(p)GVVYK; ERK-1185, GFLTEY(p)VATR]. Malachite invasion (adenoma); and tumor, invasion (adenocarcinoma). Thisgreen was obtained from BDH; Costar 96-well, half-volume, cluster protocol resulted in the development of four types of colonic tissue:culture plates were obtained from Fisher; and azoxymethane was normal colon from the control animals, and normal appearing colonicobtained from Sigma. Rabbit anti-cdc25A and horseradish peroxi- tissue, adenomas, and adenocarcinomas from the AOM-treated rats.dase-conjugated goat anti-rabbit IgG antibodies were obtained from The rats were cared for in accordance with guidelines laid down bySanta Cruz. Chemiluminescence and autoradiography films were ob- the Animal Care Committee of the University of Saskatchewan.tained from DuPont. All other chemicals and biochemicals were ob- The mitotic index (MI) was used to determine the rate of growthtained from BDH and were of Analar grade or better. in the colon epithelial cells and was determined by counting the

number of mitotic figures in 10 high power microscope fields (1400:Preparation of rat extracts. All procedures were performed at 47C,unless otherwise stated. Sprague–Dawley rats were sacrificed in a Nikon microscope) of hematoxylin and eosin-stained tissue sections.

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238 DIXON, MOYANA, AND KING

TABLE 1

Phosphotyrosyl Protein Phosphatase Substrate Specificity

Relative PTP activity (%)

Ascending colon Descending colon Smooth musclePepide

substrate Soluble Particulate Nuclear Soluble Particulate Nuclear Soluble Particulate Nuclear

src416 12 52 n.d. 12 87 n.d. 7 100 n.d.src527 30 68 n.d. 23 86 22 13 100 20cdc215 26 26 19 21 59 16 21 51 23abl393 8 39 14 5 46 11 6 55 12

Note. Dephosphorylation of phosphopeptides is expressed as percentage of the smooth muscle particulate src phosphopeptide PTP activity(src416 27.91 { 2.09 and src527 27.82 { 0.18 nmol Pi/min/mg). The 500g supernatant (first centrifugation, see Experimental Procedures) wascentrifuged at 100,000g for 1 h to yield the cytosol (supernatant) fraction. The pellet was resuspended in buffer A containing 1% (v/v) NonidetP-40, mixed for 1 h, and centrifuged at 100,000g for 1 h to yield the microsomal (supernatant) fraction. The remaining pellet was resuspendedin buffer A containing 1% (v/v) Nonidet P-40 and 600 mM KCl, mixed for 1 h, and centrifuged at 100,000g for 1 h and the resulting supernatantwas termed the cytoskeletal fraction. All cell extracts were dialyzed overnight against two changes of buffer A to remove endogenous phosphate.All subcellular fraction extracts were analyzed immediately or stored at 0807C until use. Abbreviation: n.d., not determined.

Statistical analysis was performed using the Student t test and sig- within the particulate fraction (Table 1). Particulatenificance was set at P õ 0.05. PTP activity fractionated mainly into the microsomal

Western blotting. SDS–polyacrylamide gel electrophoresis was fraction (Ç85%) versus the cytoskeletal fractionperformed with a 4% stacking and 10% resolving gel as described by

(Ç15%). Subcellular distribution of PTP activity wasKing and Sale [22]. Resolved gels were incubated in transfer bufferdependent on the substrate used; that is, certain sub-(50 mM Tris, pH 9.2, containing 40 mM glycine, 1.3 mM SDS, 20%

(v/v) methanol) for 1 h before being transferred onto nitrocellulose strates were better for certain subcellular fractionsat 0.8 mAmp/cm2 for 1 h and blocked by incubating in blocking buffer (Table 1); e.g., src416, src527, and abl393 were all excellent(100 mM Tris, pH 8, containing 150 mM NaCl, 0.05% (v/v) Tween particulate PTP substrates, while being poorer cyto-20, 3% (w/v) skimmed milk powder) for 30 min at 227C. The nitrocel-

solic PTP substrates (cytosolic PTP activity accountedlulose was incubated with anti-cdc25A (1 mg/ml) for 1–2 h at 227C,forõ15% of the total cell phosphotyrosyl abl393 peptidewashed 3 1 5 min in TBST (100 mM Tris, pH 8, containing 150

mM NaCl, 0.05% (v/v) Tween 20), then incubated with horseradish dephosphorylation). cdc215 was a good substrate for allperoxidase-conjugated anti-IgG for 1 h at 227C. The nitrocellulose cellular fractions, especially the nuclear fractions, andwas washed 4 1 5 min in TBST and the immunoreactive bands were was routinely used for most PTP assays. The onlyvisualized by chemiluminescence.

known phosphotyrosyl substrates for the cdc25 phos-phatases are phosphotyrosine 15 residues in p34cdc2

RESULTSand the CDKs, the sequences of which are conserved.Consequently, the cdc215 peptide represents a physio-Characterization of Rat Colon PTPslogically relevant phosphosubstrate for the analysis of

Colon epithelial mucosa PTP activity appeared to be the phosphotyrosyl activity of the cdc25 phosphatases.uniformly distributed down the length of the colon, ERK-1185 was a poor substrate for all cellular fractions;with similar subcellular distribution of PTP activity this may be due to a lack of a significant recognitionthrough the ascending and descending colons. The epi- sequence (the tyrosine kinase that phosphorylates thethelial cells were fractionated into nuclear and postnu- ERK-1185 tyrosine residue required the complete ERK-clear fractions and assayed for PTP activity. Ascending 1 protein for recognition).colon PTP activity divided: 3.91 { 0.05 nmol Pi/min/ Dephosphorylation of the phosphopeptides was duemg (mean { SEM, n Å 4) and 5.03 { 0.22 nmol Pi/min/ to PTP activity as sodium orthovanadate, ammoniummg (mean { SEM, n Å 39) for the postnuclear and molybdate, and ZnCl2, three classical PTP inhibitors,nuclear fractions, respectively. Descending colon PTP inhibited the enzyme activity. Vanadate inhibition wasactivity divided: 4.76 { 0.17 nmol Pi/min/mg (mean { greatest with the cytosolic PTP activity versus the par-SEM, n Å 4) and 4.46 { 0.28 nmol Pi/min/mg (mean { ticulate PTP activity. The whole nuclei required highSEM, nÅ 31) for the postnuclear and nuclear fractions, levels (ú1 mM) of sodium orthovanadate or molybdaterespectively. Similar results were obtained using either to induce significant (ú70%) in vitro PTP inhibitionthe src527 or cdc215 peptides for the postnuclear and (Table 2). High concentrations of gallium nitrate (0.8cdc215 or abl393 for the nuclear PTP assays. mM) led to a small (36%) but significant (P õ 0.05)

stimulation in the PTP activity in whole nuclei (TablePostnuclear PTP activity was located primarily

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239cdc25A IN COLON CANCER

TABLE 2 and 3.60 { 0.42 times in 53% of the descending colontumors (21 of 40) (Fig. 1). In addition, there was noEffect of Various Effectors on Descending Colonsignificant difference in the level of elevated nuclearNuclear PTP ActivityPTP in either adenomas or adenocarcinomas exhibiting

Effector Relative PTP activity increased nuclear PTP activity, i.e., 3.10 { 0.85 (n Å8) versus 3.53 { 0.41 (n Å 21) for adenoma and adeno-None 1.00carcinoma nuclear PTP activity, respectively (Fig. 2).Na3Vo4

0.5 mM 0.37 { 0.10 The expression of cdc25A was examined by Western1.0 mM 0.22 { 0.08 blotting (Fig. 3). cdc25A protein was elevated in all2.0 mM 0.11 { 0.04 individual tumors tested (numbered 1–10; consisting

EDTA of 2 ascending colon-derived tumors and 8 descending5.0 mM 1.09 { 0.08colon-derived tumors; 2 adenomas and 8 adenocarcino-Tris, pH 7.4

50 mM 1.17 { 0.07 mas) irrespective of tumor location or overall nuclearGa(NO3)3 PTP activity.

0.4 mM 1.06 { 0.140.8 mM 1.36 { 0.08 Starved and Starved/Refed Rat Colon Epithelial(NH4)6Mo7O24 PTP Activity0.5 mM 0.39 { 0.061.0 mM 0.25 { 0.08 Increased nuclear PTP activity was not due to any2.0 mM 0.17 { 0.05

change in the rates of growth of the cancerous cellNote. Whole nuclei (0.2 mg/ml) derived from the nuclear fraction (there was an increased MI in the colon tumors) as no

of normal descending colon epithelial mucosa were assayed for PTP significant change in the PTP activity occurred underactivity in the presence of cdc215 phosphopeptide (200 mM) and vari- conditions of rapid or retarded epithelial growth (seeous effectors (as indicated below) for 10–45 min. The results are below). Starvation and starvation/refeeding was useddepicted as the PTP activity relative to no addition (mean{ SEM; 4–

as a model for rapid and retarded colon epithelial cell8 experiments). Identical results were obtained for ascending colonderived nuclei. growth. In this study the rats were divided into three

groups: Group 1, the rats were allowed free access torodent chow (77% carbohydrate, 16% protein, 7% fat)throughout the experimental time period. Group 2, the2). Phosphoseryl protein phosphatase (PSP) inhibitors,rats were starved for 48 h (retarded epithelial growth).okadaic acid (up to 100 mM) and EDTA (5 mM), had noGroup 3, the rats were starved for 48 h followed byeffect on the PTP activity from any fraction of the cell.refeeding ad lib. for 24 h (rapid epithelial growth). Nu-

Tumor PTP Activity

Fifty-six colon tumors were analyzed for nuclear PTPactivity; 16 tumors were derived from the ascendingcolon and 40 tumors were derived from the descendingcolon. Ascending and descending colon tumors were ho-mogenized and nuclear and postnuclear fractions pro-duced as described under Experimental Procedures.Normal looking epithelial cells from control and AOM-injected rats were used as controls. Nuclear PTP activ-ity was analyzed by cdc215 and abl393 phosphopeptides.Tumorigenesis resulted in elevated nuclear PTP activ-ity in 52% of rat colon tumors (343.0 { 27.0% of normalepithelial PTP activity, 29 of 56 tumors, P õ 0.001)using cdc215 as substrate. No such elevation was ob-served with the postnuclear fraction. A similar increasein nuclear PTP activity was observed using the abl393

phosphopeptide as substrate, i.e., a 1.8- to 2.4-fold in-crease in the colon tumor nuclear PTP activity. There FIG. 1. Elevated nuclear PTP in ascending and descending de-

rived colon tumors. Whole nuclei (2.5 mg) from normal and tumorwas no significant difference in the proportion of tu-derived ascending and descending colon were analyzed for PTP activ-mors or the degree of elevation of PTP activity in tu-ity as described under Experimental Procedures. Elevated ascendingmors derived from either the ascending or descending or descending tumor derived nuclear PTP activity (solid) is expressed

colon; i.e., PTP activity was elevated 2.90 { 0.78 times relative to normal ascending or descending colon derived nuclearPTP activity (open).in 50% of the ascending colon derived tumors (8 of 16)

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240 DIXON, MOYANA, AND KING

FIG. 2. Nuclear PTP activity and mitotic index were determined as described under Experimental Procedures.

clear and postnuclear fractions were prepared as de- ratio than in the preinvasive adenomas. The MI of thestarved/refed colon was 10 times higher than the re-scribed above and analyzed for PTP activity. Nuclear

PTP activity under these conditions was 77.9 { 8.0% spective starved colon epithelial mucosa and not sig-nificantly different from the rates of growth observed(n Å 10 rats) and 90.7 { 7.3% (n Å 10 rats) of the

control fed animals for 48 h starved (slow epithelial in colon cancer (adenomas or adenocarcinomas).cell growth) and 48 h starved/24 h refed (rapid epithe-lial cell growth), respectively (Fig. 2, open bars). The DISCUSSIONpostnuclear activity of the same samples was 129 {12% (starved) and 137 { 13% (starved/refed) of the For a complete understanding of tyrosine phosphory-control postnuclear PTP activity, respectively. Tissue lation it is necessary to study both the ‘‘on’’ (kinase) andsections from these rats were stained with hematoxylin ‘‘off’’ (phosphatase) reactions. This is especially true inand eosin as described for the colon tumors (see above) situations where aberrant tyrosine phosphorylation isand the MI for each section determined (Fig. 2, solid known to cause altered cell growth, as is seen in a vari-bars). MI values were: (1) normal colon (32.5 { 5.5), (2) ety of cancers. Such is the case with the colon. For exam-normal appearing colon from AOM-treated rats (48.5{ ple, a common early event in colon cancer is increased15.5; P Å not significant), (3) adenomas (164.3 { 27.1; activity of the c-src tyrosine kinase activity. Src activityP õ 0.05), (4) adenocarcinomas (145.0 { 12.0; P õ is also elevated in idiopathic ulcerative colitis [21], and0.001), (5) starved colon (11.5 { 2.1; P õ 0.05), and (6) the latter disease is a known risk factor for colon cancer.starved/refed colon (114.0 { 15.0; P õ 0.01). In the Indeed patients with ulcerative colitis frequently de-invasive lesions there was a pronounced desmoplastic velop colon cancer 10 years earlier than the nationaltissue response resulting in a lower epithelial/stromal average (this is especially true in long-standing cases

with extreme bowel inflammation). In addition to thisSAP-1, a receptor PTP has been observed to be highlyexpressed in colon cancer cells but not in normal colonepithelial cells [12]. The present work has studied theexpression of a potential protooncogene, cdc25A, and nu-clear PTP activity in normal colon and in colon cancer.A rat AOM system was used as it offered the best physio-logically relevant model that could be manipulated.Whole nuclei possess large pores (Ç90 nm in diameter)that are accessible to molecules of molecular massFIG. 3. Expression of cdc25A in normal and tumor derived colonõ50,000 Da and are freely permeable to small moleculesnuclei. Whole nuclei (10 mg) were electrophoresed, transferred, blot-

ted with cdc25A (1 mg/ml), and visualized as described under Experi- (õ5000 Da). The molecular mass of any of the assayment Procedures. Lane AC, normal ascending colon nuclei; lane DC, reagents is õ1500 Da, consequently while no hinder-normal descending colon nuclei; lanes 1–5 and 8–10, descending ance from diffusion will occur using the whole nucleicolon tumor derived nuclei; lanes 6, 7, ascending colon tumor derived

versus a nuclei extract, the physiological nuclear struc-nuclei. The relative PTP activities of each nuclei sample are indicated(performed in triplicate). ture and protein interactions are maintained.

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241cdc25A IN COLON CANCER

Rat colon epithelial mucosa possess high PTP activ- and 7) and lane DC with tumors 1–5 (lanes 1–5) andtumors 8–10 (lanes 8–10). The levels of cdc25A expres-ity as compared with other rat tissues, i.e., liver, mus-

cle, and adipose tissue [22]. Dephosphorylation of phos- sion were approximately consistent with the observedincreases in the PTP activities within these tumorsphopeptides was linear to ¢30%; however, to ensure

that all dephosphorylation reactions were linear, nor- (mean increase of 3.43 { 0.37) if the heterogeneity ofthe tumors is accounted for; the colon tumors consistmal PTP assays were restricted toõ30% dephosphory-

lation. Multiple phosphotyrosine phosphopeptides of a mixture of cancerous, normal, and inflammatorycells. Consequently the actual PTP in the tumor cellswere chosen for use as substrates for the various sub-

cellular fractions based on their subcellular localiza- is likely to be far greater than the gross PTP activitiesindicate. These results are consistent with those re-tion. For example, c-src and c-abl are localized to the

plasma membrane and were selected for assaying par- ported by Galaktionov et al. [5] who observed elevatedcdc25A and cdc25B expression in human breast cancer.ticulate PTP activity. The phosphopeptides demon-

strated a degree of subcellular selectivity (albeit small); To date the analysis of 56 individual colon tumors hasshown that nuclear PTP activity was elevated in ap-i.e., there was 5–9 times more abl PTP activity in the

particulate fraction than the cytosolic fraction (Table proximately 52% of cases with no significant variationin PTP activity from tumors derived from the ascending1). Unlike many other PTPs, the cdc25 phosphatases

possess a narrow substrate specificity with the phos- colon versus the descending colon (Fig. 1). In additionto this, the elevation of nuclear PTP activity appearsphotyrosyl residue 15 in p34cdc2 and the CDK cell cycle

kinases as the only known physiological substrates. to be an early event as no significant differences wereobserved between the nuclear activities derived fromTherefore, the cdc215 phosphotyrosyl peptide was cho-

sen to assay the nuclear PTP activity to ensure that adenomas versus adenocarcinomas (Fig. 2). cdc25Apossesses two apparently paradoxical functions. Thecdc25 phosphatase activity was analyzed. Clearly, even

in a relatively simple system such as isolated nuclei first is as a control for cell cycle progression: blockingthe expression of cdc25A can prevent the cell from pro-several different PTPs will be active and able to dephos-

phorylate the cdc15 phosphopeptide. However, despite ceeding to S phase and entering the cell cycle. Thus itis easy to see that an overexpression of cdc25A wouldthis problem whole nuclei possess a distinct advantage

over isolated enzyme systems in that the nuclei retains act to increase the proportion of cells in mitosis, as seenin cancer. This scenario may explain the observationsmuch of its physiological structure, maintaining pro-

tein–protein interactions that may be important in seen in this work: three- to fivefold increase in the MIand elevated cdc25A protein expression in colon cancer;PTP activity.

Dephosphorylation activity from all fractions of the that is, a greater proportion of the cells in the tumorwere undergoing mitosis compared with the normal ep-epithelial cell was sensitive to the PTP inhibitors so-

dium vanadate, zinc chloride, and ammonium molyb- ithelial mucosa. For this reason it was possible thatthe changes in the nuclear PTP activity may simplydate, indicating that PTP activity was being assayed.

Minimal dephosphorylation could be accounted for by represent changes in the rate of cellular growth. How-ever, clearly elevated growth rates are not alone suffi-PSPs as both okadaic acid and EDTA had no effect

on the dephosphorylation reaction; the phosphotyrosyl cient to elevate nuclear PTP activity as no such in-crease occurred in the model of rapid and retarded epi-activity of the type 2 PSPs have an absolute require-

ment for divalent metal ions [22, 23]. Gallium nitrate thelial mucosa growth [24]; i.e., there was no differencein the PTP activity derived from colon nuclei undermildly stimulated the nuclear PTP activity at high (800

mM) but not lower (400 mM) concentrations. The rea- conditions of fast (starvation/refeeding: three- to five-fold increase in MI) and slow (starvation: 65% decreasesons for this are unclear although this result is not

inconsistent with the results of Berggren et al. [16] who in MI) epithelial cell growth. Thus the changes re-ported here represent an aspect of tumorigenesis. Italso found no effect on cdc25 activity at low (100 mM)

concentrations of gallium nitrate. will be important to determine whether the changesare primary, and may potentially be used to manipu-The analysis of the levels of cdc25A protein by West-

ern blotting indicated that the expression of cdc25A late the growth of the tumor, or are secondary, theresults of other cellular changes, and also whether thewas greater in the mucosa derived from normal de-

scending colon compared with normal ascending colon change in nuclear PTP activity can be accounted forsolely by changes in cdc25A expression and/or activity.(Fig. 3, lanes DC and AC). Nuclei isolated from individ-

ual tumors by Western blotting indicated that cdc25A The activities of the cdc25 phosphatases are them-selves regulated by protein phosphorylation/dephos-expression was elevated in all tumors (tumors are num-

bered 1–10). The corresponding elevation levels of phorylation. The increase in cdc25A expression com-pares favorably with many of the classical oncogenes,cdc25A protein ranged from approximately two- to six-

fold; compare lane AC with tumors 6 and 7 (lanes 6 where typically 30% of breast cancers may express erb

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242 DIXON, MOYANA, AND KING

5. Galaktionov, K., Lee, A. K., Eckstein, J., Draetta, G., Meckler,B2 [15–17]. The second cdc25A function is in the con-J., Loda, M., and Beach, D. (1995). CDC25 phosphatases astrol of myc-dependent apoptosis; where c-myc can di-potential human oncogenes. Science 269, 1575–1577.rectly stimulate the synthesis of cdc25A and under con-

6. Klarland, J. K. (1985). Transformation of cells by an inhibitorditions of serum starvation cause apoptosis. Again, of phosphatases acting on phosphotyrosine in proteins. Cell 41,blocking the production of cdc25A (with antisense olio- 707–717.gonucleotides) resulted in an inhibition of c-myc in- 7. Gentleman, S., Martensen, T. M., Digiovanna, J. J., and

Chader, G. J. (1984). Protein tyrosine kinase and protein phos-duced apoptosis [25].photyrosine phosphatase in normal and psoriatic skin. Biochim.At present very little work has been undertaken in-Biophys. Acta 798, 53–59.volving the role of PTPs in tumorigenesis. However,

8. Wiener, J. R., Kerns, B.-J. M., Harvey, E. L., Conaway, M. R.,such work that has been published has shown signifi-Iglehart, C. J. D., Berchuck, A., and Bast, R. C. (1994). Overex-cant effects with cell transformation and tumorigene- pression of the protein tyrosine phosphatase PTP1B in human

sis. Whether these changes are part of the pathogenesis breast cancer: Association with p185c-erbB-2 protein expres-sion. J. Natl. Cancer Inst. 86, 372–378.of cancer or a mechanism whereby the cell attempts to

9. Kidd, K. R., Kerns, B.-J. O., Dodge, R. K., and Wiener, J. R.curtail and control elevated tyrosine kinase activity has(1992). Histochemical staining of protein-tyrosine phosphatasestill to be determined. To date, no other study has beenactivity in primary human mammary carcinoma: Relationshipundertaken to investigate PTPs in primary colon can-with established prognostic indicators. J. Histochem. Cytochem.

cer. Several groups have used colon cancer cell lines 40, 729–735.for study; however, as cancer cell lines do not represent 10. Wiener, J. R., Hurteau, J. A., Kerns, B.-J. M., Whitaker, R. S.,a physiological system, care should be used in the inter- Conaway, M. R., Berchuck, A., and Bast, R. C. (1994). Overex-

pression of the tyrosine phosphatase PTP1B is associated withpretation of the results. The process of immortalizationhuman ovarian carcinomas. Am. J. Obstet. Gynecol. 170, 1177–results in a dramatic alteration in the phosphorylation1183.kinetics [26] indicating a potential change in the kinase

11. Zhai, Y. F., Beittenmiller, H., Wang, B., Gould, M. N., Oakley,and phosphatase activities.C., Esselman, W. J., and Welsch, C. W. (1993). Increased ex-

Before 1990 the number of PTPs in a cell was thought pression of specific protein tyrosine phosphatases in humanto be limited, with few PTPs doing many overlapping breast epithelial cells neoplastically transformed by the neu

oncogene. Cancer Res. 53, 2272–2278.and largely uncontrolled activities (it was thought that12. Matozaki, T., Suzuki, T., Uchida, T., Inazawa, J., Ariyama, T.,the kinase reaction represented the controlled biologi-

Matsuda, K., Horita, K., Noguchi, H., Mizuno, H., Sakamoto,cal pathway). With the work of Tonks and others theC., and Kasuga, M. (1994). Molecular cloning of a human trans-number of PTPs and complexity of the dephosphoryla-membrane-type protein tyrosine phosphatase and its expres-

tion reaction are becoming evident, with the data gen- sion in gastrointestinal cancers. J. Biol. Chem. 269, 2075–2081.erated from the sequencing of the human genome pre- 13. Lammers, R., Bossenmaier, B., Cool, D. E., Tonks, N. K., Schles-dicting approximately 500 PTPs [27]. In light of recent singer, J., Fischer, E. H., and Ullrich, A. (1993). Differential

activities of protein tyrosine phosphatases in intact cells. J.results, the complex and conflicting roles of PTPs areBiol. Chem. 268, 22,456–22,462.becoming more and more evident and thus demand

14. Brown-Shimer, S., Johnson, K. A., Hill, D. E., and Bruskin,intense study in the future.A. M. (1992). Effect of protein tyrosine phosphatase 1B expres-sion on transformation by the human neu oncogene. Cancer

We are grateful for the help of Dr. A. Magliocco (Department of Res. 52, 478–482.Pathology, University of Saskatchewan) for his assistance in grading 15. Ramponi, G., Ruggiero, M., Raugei, G., Berti, A., Modesti, A.,the colon tumors and the Immunohistology Laboratory (Department Degl’Innocent, D., Magnelli, L., Pazzagli, C., Chiarugi, V. P.,of Pathology, Royal University Hospital) for the processing of the and Camici, G. (1992). Overexpression of a synthetic phospho-tissues. This work was supported by grants from NCIC (Terry Fox tyrosine protein phosphatase gene inhibits normal and trans-regional development grant) and the Saskatchewan Health Services formed cell growth. Int. J. Cancer 51, 652–656.Utilization and Research Commission.

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Received November 24, 1997

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