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EXPRESSION OF PROTEIN KINASE C b1 CONFERS RESISTANCE TO TNFa- AND PACLITAXEL-INDUCED APOPTOSIS IN HT-29 COLON CARCINOMA CELLS Patrizia CESARO 1 , Elisabetta RAITERI 1 , Marina D´ EMOZ 1 , Roberta CASTINO 1 , Francesco M. BACCINO 2,3 , Gabriella BONELLI 2 and Ciro ISIDORO 1 * 1 Dipartimento di Scienze Mediche, Universita ` “A. Avogadro”, Novara, Italy 2 Dipartimento di Medicina ed Oncologia Sperimentale, Universita ` di Torino, Torino, Italy 3 Centro CNR di Immunogenetica ed Oncologia Sperimentale, Torino, Italy The expression of different protein kinase C (PKC) isoen- zymes has been shown to vary with proliferation rates, dif- ferentiation or apoptosis in normal colon crypts. In addition, the activity of some PKC isoenzymes appears to be reduced in colorectal cancer. The aim of the present work was to determine whether modulation of PKC expression would affect the susceptibility of a p53-defective colon carcinoma cell line to different apoptotic treatments. HT-29 cells exhib- ited sensitivity to paclitaxel (Taxol) and tumor necrosis fac- tor a (TNFa) in a dose- and time-dependent manner but were relatively resistant to etoposide. Inhibition of PKC ac- tivity augmented the susceptibility of HT-29 cells to apopto- sis, and phorbol ester induction of PKC reduced such suscep- tibility. Transfected HT-29 PKC cells, hyper-expressing the b1 isoform of PKC, were less sensitive to TNFa and paclitaxel than the normal counterpart. The present data 1) indicate that the expression of PKC influences the susceptibility of HT-29 colon cancer cells to apoptotic drugs apparently re- gardless of their mechanism of action, and 2) suggest pacli- taxel as a potential candidate for the treatment of colon cancer, possibly in association with inhibitors of PKC (a and b) at doses not cytotoxic per se. © 2001 Wiley-Liss, Inc. Key words: protein kinase C; TNFa; paclitaxel; apoptosis; colon carcinoma Colorectal cancer is one of the most common solid tumors world-wide. Due to its high metastatic potential and the frequent onset of resistance to chemotherapy, it is one of the four major causes of death by neoplasia in westernized countries. Loss of function of p53 occurs in more than 75% of human colorectal cancers. Since p53 regulates a complex array of cellular responses to DNA damage, including cell cycle arrest and apoptosis, its loss of function is also expected to affect the sensitivity of tumor cells to DNA-damaging antiblastic drugs. 1,2 The expression of different protein kinase C (PKC) isoenzymes varies with proliferation rates, differentiation or apoptosis in nor- mal colon crypts, 3–6 and the activity of some PKC isoenzymes is reduced in colorectal cancer. 7–9 Whether, in addition to loss of function of p53, altered functioning of PKCs contributes to the low sensitivity of colon carcinomas to apoptosis-based chemotherapy remains to be established. In the present work we addressed this issue by examining the effect of various apoptotic treatments on HT-29 colon cancer cells that lack functional p53 10 and either do or do not express abnormal levels of the isoform b1 of PKC. We found that paclitaxel (Taxol) and TNFa, but not etoposide, effi- ciently induced apoptosis of HT-29 cells in a dose- and time- dependent manner. The involvement of PKC in the apoptotic pathways triggered by paclitaxel or TNFa was assessed by using a myristoylated pseudo- substrate as inhibitor at doses not affecting cell viability. In HT-29 cells, inhibition of PKC (a and b isoforms) activity augmented by twofold the cytotoxicity of both drugs. Induction of PKC by the phorbol ester phorbol myristate acetate (PMA) decreased the sus- ceptibility of HT-29 cells to both TNFa and paclitaxel treatments. Resistance to apoptotic treatments was consistently observed in transfected HT-29 cells hyper-expressing PKCb1. The present data implicate PKC (a and b) as a component of the mechanism responsible for the resistance of colon carcinoma cells to apoptotic drugs. MATERIAL AND METHODS Cells and chemicals The HT-29 human colon cancer cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD). HT- 29 PKC (clone 7) and HT-29C1 cells 11 derive from HT-29 cells transfected with the cDNA encoding PKCb1 or with the vector lacking the PKC DNA insert, respectively, and were kindly pro- vided by Dr. Weinstein (Columbia University, New York, NY). Tissue culture medium and antibiotics were purchased from Sigma (St. Louis, MO), and FCS was from Gibco (Gaithersburg, MD). Human recombinant TNFa was obtained from REB (Abingdon Oxon, UK), and paclitaxel was purchased from Bristol-Myers Squibb (Sermoneta, LT, Italy). The myristoylated PKC-a and -b pseudo-substrate (inhibitor of PKC) was purchased from BACHEM (Bubendorf, Switzerland). Etoposide (VP-16), PMA, 4-6-diamidino-2-phenyl-indol-dihydrochloride (DAPI), propidium iodide and other chemicals were from Sigma. Western blotting analysis of PKCb1 expression Analysis of PKCb1 expression was performed by standard Western blotting. Briefly, 50 mg of cell homogenates were frag- mented by electrophoresis on a 12.5% polyacrylamide gel and electroblotted onto nitrocellulose. PKCb1 was detected by chemi- luminescence reaction on a filter decorated with a specific poly- clonal rabbit antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) followed by horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Richmond, CA). A gel was run in parallel and stained with Coomassie Blue to verify equal loading of sam- ples. The amount of b-actin per sample loaded was determined by re-probing the same filter with polyclonal anti-b-actin antibodies (Sigma). Bands on films were quantitated by densitometry (Bio- Grant sponsor: the Ministero dell’Universit` a e della Ricerca Scientifica e Tecnologica (Roma, cofin. 1997 and 1998); Grant sponsor: the Associa- zione Italiana per la Ricerca sul Cancro (AIRC, Milano); Grant sponsor: the Consiglio Nazionale delle Ricerche (CNR, target project on Biotech- nology; Grant number: 9900386PF.49; Grant sponsor: the Universit` a del Piemonte Orientale “A. Avogadro”; Grant sponsor: the Fondazione Cava- lieri Ottolenghi (Torino). Patrizia Cesaro and Elisabetta Raiteri contributed equally to this work. *Correspondence to: Dipartimento di Scienze Mediche, Universit` a “Amedeo Avogadro”, Via Solaroli 17, 28100 Novara, Italy. Fax: 139-0321-620421. E-mail: [email protected] Received 28 July 2000; Revised 9 January 2001; Accepted 9 February 2001 Published online 11 April 2001 Int. J. Cancer: 93, 179 –184 (2001) © 2001 Wiley-Liss, Inc. Publication of the International Union Against Cancer

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EXPRESSION OF PROTEIN KINASE C b1 CONFERS RESISTANCETO TNFa- AND PACLITAXEL-INDUCED APOPTOSIS IN HT-29COLON CARCINOMA CELLSPatrizia CESARO

1, Elisabetta RAITERI1, Marina DEMOZ

1, Roberta CASTINO1, Francesco M. BACCINO

2,3, Gabriella BONELLI2 and

Ciro ISIDORO1*

1Dipartimento di Scienze Mediche, Universita “A . Avogadro”, Novara, Italy2Dipartimento di Medicina ed Oncologia Sperimentale, Universita di Torino, Torino, Italy3Centro CNR di Immunogenetica ed Oncologia Sperimentale, Torino, Italy

The expression of different protein kinase C (PKC) isoen-zymes has been shown to vary with proliferation rates, dif-ferentiation or apoptosis in normal colon crypts. In addition,the activity of some PKC isoenzymes appears to be reducedin colorectal cancer. The aim of the present work was todetermine whether modulation of PKC expression wouldaffect the susceptibility of a p53-defective colon carcinomacell line to different apoptotic treatments. HT-29 cells exhib-ited sensitivity to paclitaxel (Taxol) and tumor necrosis fac-tor a (TNFa) in a dose- and time-dependent manner butwere relatively resistant to etoposide. Inhibition of PKC ac-tivity augmented the susceptibility of HT-29 cells to apopto-sis, and phorbol ester induction of PKC reduced such suscep-tibility. Transfected HT-29PKC cells, hyper-expressing the b1isoform of PKC, were less sensitive to TNFa and paclitaxelthan the normal counterpart. The present data 1) indicatethat the expression of PKC influences the susceptibility ofHT-29 colon cancer cells to apoptotic drugs apparently re-gardless of their mechanism of action, and 2) suggest pacli-taxel as a potential candidate for the treatment of coloncancer, possibly in association with inhibitors of PKC (a andb) at doses not cytotoxic per se.© 2001 Wiley-Liss, Inc.

Key words: protein kinase C; TNFa; paclitaxel; apoptosis; coloncarcinoma

Colorectal cancer is one of the most common solid tumorsworld-wide. Due to its high metastatic potential and the frequentonset of resistance to chemotherapy, it is one of the four majorcauses of death by neoplasia in westernized countries. Loss offunction of p53 occurs in more than 75% of human colorectalcancers. Since p53 regulates acomplex array of cellular responsesto DNA damage, including cell cycle arrest and apoptosis, its lossof function is also expected to affect the sensitivity of tumor cellsto DNA-damaging antiblastic drugs.1,2

The expression of different protein kinase C(PKC) isoenzymesvaries with proliferation rates, differentiation or apoptosis in nor-mal colon crypts,3–6 and the activity of some PKC isoenzymes isreduced in colorectal cancer.7–9 Whether, in addition to loss offunction of p53, altered functioning of PKCscontributes to the lowsensitivity of colon carcinomas to apoptosis-based chemotherapyremains to be established. In the present work we addressed thisissue by examining the effect of various apoptotic treatments onHT-29 colon cancer cells that lack functional p5310 and either door do not express abnormal levels of the isoform b1 of PKC. Wefound that paclitaxel (Taxol) and TNFa, but not etoposide, effi-ciently induced apoptosis of HT-29 cells in a dose- and time-dependent manner.

The involvement of PKC in theapoptotic pathways triggered bypaclitaxel or TNFa wasassessed by using amyristoylated pseudo-substrate as inhibitor at doses not affecting cell viability. In HT-29cells, inhibition of PKC (a and b isoforms) activity augmented bytwofold the cytotoxicity of both drugs. Induction of PKC by thephorbol ester phorbol myristate acetate (PMA) decreased the sus-ceptibility of HT-29 cells to both TNFa and paclitaxel treatments.Resistance to apoptotic treatments was consistently observed intransfected HT-29 cells hyper-expressing PKCb1. The present

data implicate PKC (a and b) as acomponent of the mechanismresponsible for the resistanceof colon carcinomacells to apoptoticdrugs.

MATERIAL AND METHODS

Cells and chemicalsThe HT-29 human colon cancer cell line was obtained from the

American Type Culture Collection (ATCC, Rockville, MD). HT-29PKC (clone 7) and HT-29C1 cells11 derive from HT-29 cellstransfected with the cDNA encoding PKCb1 or with the vectorlacking the PKC DNA insert, respectively, and were kindly pro-vided by Dr. Weinstein (Columbia University, New York, NY).Tissueculturemedium and antibioticswerepurchased from Sigma(St. Louis, MO), and FCS was from Gibco (Gaithersburg, MD).Human recombinant TNFa was obtained from REB (AbingdonOxon, UK), and paclitaxel was purchased from Bristol-MyersSquibb (Sermoneta, LT, Italy). The myristoylated PKC-a and -bpseudo-substrate (inhibitor of PKC) was purchased fromBACHEM (Bubendorf, Switzerland). Etoposide (VP-16), PMA,4-6-diamidino-2-phenyl-indol-dihydrochloride (DAPI), propidiumiodide and other chemicals were from Sigma.

Western blotting analysis of PKCb1 expressionAnalysis of PKCb1 expression was performed by standard

Western blotting. Briefly, 50 mg of cell homogenates were frag-mented by electrophoresis on a 12.5% polyacrylamide gel andelectroblotted onto nitrocellulose. PKCb1 was detected by chemi-luminescence reaction on a filter decorated with a specific poly-clonal rabbit antiserum (Santa Cruz Biotechnology, Santa Cruz,CA) followed by horseradish peroxidase-conjugated secondaryantibodies (Bio-Rad, Richmond, CA). A gel was run in paralleland stained with Coomassie Blue to verify equal loading of sam-ples. The amount of b-actin per sample loaded was determined byre-probing the same filter with polyclonal anti-b-actin antibodies(Sigma). Bands on films were quantitated by densitometry (Bio-

Grant sponsor: the Ministero dell’Universita edella Ricerca Scientificae Tecnologica (Roma, cofin. 1997 and 1998); Grant sponsor: the Associa-zione Italiana per la Ricerca sul Cancro (AIRC, Milano); Grant sponsor:the Consiglio Nazionale delle Ricerche (CNR, target project on Biotech-nology; Grant number: 9900386PF.49; Grant sponsor: the Universita delPiemonte Orientale “A . Avogadro”; Grant sponsor: the Fondazione Cava-lieri Ottolenghi (Torino).

Patrizia Cesaro and Elisabetta Raiteri contributed equally to this work.

*Correspondence to: Dipartimento di Scienze Mediche, Universita“Amedeo Avogadro”, Via Solaroli 17, 28100 Novara, Italy.Fax: 139-0321-620421. E-mail: [email protected]

Received 28 July 2000; Revised 9 January 2001; Accepted 9 February2001

Published online 11 April 2001

Int. J. Cancer: 93, 179–184 (2001)© 2001 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

Rad GelDoc 1,000; software Quantity One 4.03). Pre-stained stan-dard molecular weight proteins were from Bio-Rad.

Cell culture and treatmentsHT-29, HT-29C1 and HT-29PKC cells were grown under stan-

dard culture conditions in DMEM with antibiotics and 10% heat-inactivated FCS as previously reported.12 G418 (Sigma) was in-cluded as the selecting antibiotic in the culture medium oftransfected cells. For the experiments, HT-29 cells were plated in25 cm2 flasks at 53 104 cells/cm2 initial density and left to adherefor 48 hr prior to treatment. By this time cell density had reacheda value of approximately 123 104 cells/cm.2 In serum-fed HT-29cultures, cell density was approximately 253 104 cells/cm2 after24 hr and 403 104 cells/cm2 after 48 hr. In this respect, HT-29C1behaved like the parental HT-29 cell line. Since the growth rate ofHT-29PKC cells is lower than that of HT-29, HT-29PKC cells wereplated at 83 104 cells/cm2 the initial density so that both cell lineshad a comparable cell density by the time the cytotoxic treatmentswere applied. Treatments lasted for 24 or 48 hr. In the latter casethe medium was renewed after the first 24 hr in order to avoid sideeffects from nutrient consumption and accumulation of toxic me-tabolites; the drug was then re-added.

Evaluation of cytotoxic effectsAt the times indicated, control and treated cultures were

trypsinized, and the cells were counted. The percentage of adher-ent viable cells was determined by counting the trypan blue-excluding cells with a hemocytometer. For a rough evaluation ofmorphology and cell density and for detection of apoptotic ornecrotic cells, cultures were observed at 12 hr intervals under aphase-contrast microscope and photographed. The appearance of ahypodiploid (sub-G1) cell population, corresponding to apoptoticcells, was monitored by flow cytometry on whole cell populations(i.e., monolayer plus cells recovered from the medium) fixed andstained with propidium iodide as described previously.13 Chroma-tin alterations were revealed in fixed cell monolayers by DNAfluorescent staining with DAPI (1mg/ml methanol for 30 min),observed under a UV microscope and photographed.

RESULTS

Apoptosis of HT-29 cells in response to TNFa or paclitaxel isdose and time dependent

Allelic deletion or inactivating mutations of the p53 tumorsuppressor gene are well documented in colorectal carcinomas.Disruption of p53 function has profound cellular consequencesincluding reduced sensitivity to apoptotic treatments.1,2,14 Weevaluated HT-29 colorectal cancer cells, lacking functional p53,10

for their sensitivity to apoptotic drugs that act at different cellularlevels and are of potential interest for the treatment of this cancer.For this purpose we incubated HT-29 cells with the antiblasticdrugs etoposide (VP-16) or paclitaxel or with the cytokine TNFa.Cells were exposed to various concentrations of the cytotoxicdrugs for periods ranging from 4 to 48 hr. To ensure optimalconditions for cell growth, the medium was renewed after 24 hr ofculture, and, where indicated, the cytotoxic substances were re-added. At the end of the incubation, surviving adherent cells werecounted. The occurrence of cell death by apoptosis was ascertainedby cytofluorometric analysis of propidium iodide-labeled DNAand DAPI decoration of nuclei.

Some significant results of such treatments, expressed as per-centage of cell survival in treated cultures compared with controls,are summarized in Table I.Paclitaxel only exerted a cytotoxiceffect within the first 16 hr of treatment. Etoposide, at a concen-tration shown to be effective on many cell types,13,15,16displayeda low cytotoxicity on HT-29 cells treated for as long as 48 hr. Ahigher rate of apoptosis was obtained by treating HT-29 cells for48 hr with 100 ng/ml TNFa, a rather high concentration comparedwith those sufficient to induce apoptosis in other tumor celltypes17–19 (Table I). Cytofluorometric analysis of cell cycle dis-

tribution (data not shown) indicated that TNFa initially inducedgrowth arrest (first 24 hr of treatment) and later also induced celldeath, which became apparent by 48 hr. Paclitaxel was a quitepowerful apoptogenic agent for HT-29 cells since it was effectiveat a concentration as low as 20 nM (data not shown). In culturestreated with 5mM paclitaxel, about 30% of cells underwentapoptosis within 4 hr, and more than 65% of cells underwentapoptosis by 24 hr (data not shown). With 50 nM of paclitaxel, cellsurvival accounted to 57% of control by 24 hr and this percentagedecreased to about 12% by 48 hr (Table I). With 500 nM ofpaclitaxel, cell viability was even more drastically reduced at anytime.

In a separate set of experiments the treatment was prolonged to48 hr in unchanged medium and without re-adding the substances.In these experimental conditions cytotoxicity by TNFa and byetoposide increased, although in the case of the latter, necrosis wasalso observed. Because of its low apoptogenic efficacy on HT-29cells, etoposide was not used further.

We examined TNFa- and paclitaxel-treated cultures for mor-phological detection of apoptosis by DAPI staining. When exposedto 100 ng/ml TNFa, HT-29 became larger in size and showedcytoplasmic extension and intracytoplasmic vacuoles within thefirst 24 hr; chromatin condensation and formation of DNA-con-taining apoptotic bodies became evident by 48 hr of treatment (Fig,1). Twenty-four hours after exposure to 50 nM paclitaxel, HT-29cells became smaller and rounded, displaying typical apoptoticfeatures, such as chromatin condensation and fragmentation ofnuclei into small spherical particles (Fig. 1).

In the following experiments, we explored the role of PKC inthe cellular response of HT-29 cells to TNFa and paclitaxel.Because of their different efficacies, we used TNFa and paclitaxelunder experimental conditions that caused a comparable cytotoxiceffect on the cells (50 nM paclitaxel for 24 hr and 100 ng/ml TNFafor 48 hr).

Modulation of classical protein kinase C activity alters thesensitivity to cytotoxic agents

PKC serves as second messenger in pathways signaled bysurvival, growth and pro-differentiative factors.20,21 Inhibition ofPKC activity by staurosporine or its derivatives results in cell cycleblock and eventually apoptosis in HT-29 cells.22,23 We wonderedwhether modulation of PKC activity would impact the sensitivityof HT-29 colon carcinoma cells to TNFa or paclitaxel. In a first setof experiments, we inhibited PKC activity by using a cell-perme-able myristoylated pseudo-substrate that selectively inhibits theisoformsa andb of PKC.24 We assessed the optimal concentrationof this drug (hereafter referred to as inhibitor of PKC [IPKC]) thatdid not affect the growth of the cultures for as long as 72 hr. Asshown in Figure 2, inhibition of PKC activity increased by nearlytwofold the sensitivity of HT-29 cells to both the apoptotic drugsused. This confirms and extends previous data23 suggesting that inp53-deficient colon carcinoma cells PKC (specificallya and b

TABLE I – EFFECTS OF CYTOTOXIC TREATMENTS ON HT-29 CELLMONOLAYERS1

time (h)VP-16 (mM) TNFa (ng/ml) Paclitaxel (nM)

1 10 50 100 50 500

6 100 100 100 100 100 10016 100 92 100 100 886 9 776 924 100 786 9 856 7 886 4 576 4 256 448 906 9 706 8 786 5 546 6 126 4 46 21Adherent HT-29 cells were exposed to different apoptotic treat-

ments as indicated. Cell growth at the various time points was deter-mined by counting viable adherent cells. Data from four separateexperiments in duplicate report the percentage of cell survival intreated cultures compared with controls. (S.D. is not reported fortreatments that revealed no cytotoxicity.) TNFa, tumor necrosis factora.

180 CESAROET AL.

isoforms) is also a central molecule in pathways that promote cellsurvival and counteract apoptosis.

To investigate this issue further, we examined the sensitivity toTNFa or paclitaxel of HT-29 cells (pre)-treated with PMA underconditions that stimulated intracellular PKC activity. Experimentalconditions (PMA concentration, absence of serum and time ofincubation) were assessed based on data from the literature25 andon our own experience (unpublished data). In the present experi-ment, serum was omitted to avoid activation of PKC by otherexogenous stimuli. In cultures treated for as long as 72 hr with 50nM PMA, cell growth was slowed, but apoptotic death was notapparent. As shown in Figure 3a, the percentage of cell survivalafter 48 hr of incubation with TNFa approached 57% in PMA-untreated cells, 75% in cultures pre-treated for 24 hr with PMAand 87% in cultures in which PMA was present for 24 hr prior toand 48 hr throughout the incubation with the cytokine. Parallelexperiments were performed in which paclitaxel was employed inthe last 24 hr of incubation. Survival under paclitaxel treatmentincreased to about 80% in cultures exposed to PMA only for thefirst 24 hr and reached nearly 100% in cultures exposed to PMA

for the whole experimental period (Fig. 3b). Therefore, stimulationof PKC by PMA, which mainly acts ona andb isoforms, appearsto render HT-29 cells more resistant to TNFa and to paclitaxelaction.

Hyper-expression of PKCb1 modifies the response of HT-29colorectal cancer cells to apoptotic treatments

We wished to clarify further the role of classical PKCs in HT-29cells and therefore focused our attention on theb isoform, takingadvantage of the fact that HT-29 cells transfected with the cDNAencoding PKCb1 and hyper-expressing this PKC isoform11 wereavailable in our laboratory (kindly provided by Dr. Weinstein). Weexamined the sensitivity of the transfected HT-29PKC cells toTNFa or paclitaxel. HT-29C1 cells (the parental clone transfectedwith the empty vector) were shown to be indistinguishable fromthe parental untransfected HT-29 cells with respect to morphology,growth characteristic (duplication time, serum requirements, satu-ration density), PKC levels and response to PMA (data not shown

FIGURE 1 – Tumor necrosis factora (TNFa) and paclitaxel (Taxol[Tax]) alter chromatin organization in HT-29 cells. HT-29 cells weregrown on sterile coverslips and treated with cytotoxic agents (100ng/ml TNFa for 48 hr or 50 nM paclitaxel for 24 hr). Cells were thenfixed and stained with 0.1 mg/ml DAPI for 30 min and observed andphotographed under the UV microscope. In TNFa-treated culturesnuclear fragments included in apoptotic bodies are clearly visible. Inpaclitaxel-treated cultures, most cells undergo apoptosis, as detectedby chromatin condensation and nuclear fragmentation. Co, control.(Original magnification31,000.)

FIGURE 2 – Inhibition of protein kinase C (PKC)a and b isoen-zymes and susceptibility of HT-29 cells to tumor necrosis factora(TNFa)- or paclitaxel (Taxol [Tax])-induced apoptosis. (a) Culturesincubated for 48 hr in the absence (control [Co]) or presence of 100mM myristoylated pseudo-substrate ofa andb PKC isoforms (IPKC),or 100 ng/ml TNFa or a combination of both as indicated. (b) Culturesincubated for 24 hr in the absence (Co) or presence of 100mMmyristoylated pseudo-substrate ofa andb PKC isoforms (IPKC), or 50nM paclitaxel or a combination of both as indicated. The number ofviable cells in treated cultures is calculated as percentage of controls.Data represent the mean6 SD of three independent experiments intriplicate. At the concentration used in the present experiments, theinhibitor of PKC did not affect cell viability but strongly potentiatedthe cytotoxicity by TNFa and paclitaxel.

181ROLE OF PKC IN COLON CARCINOMA CELL DEATH

and Fig. 4; see also ref. 26). Transfection of PKCb1 resulted in anearly 15-fold increase in PKC activity.11

We analyzed by Western blotting the expression of the PKCb1protein in HT-29 cells and in the clones HT-29C1 and HT-29PKC.As shown in Figure 4, HT-29 and HT-29C1 cells expressed equalamounts of the protein, whereas HT-29PKC expressed a muchhigher level of the protein, as expected. Given the similarity ofHT-29 and HT-29C1 with respect to growth characteristics andPKC expression, for the sake of homogeneity in the followingexperiment we utilized the parental HT-29 cell line as the coun-terpart for HT-29PKC. When exposed to apoptotic treatments,HT-29PKC cells were more resistant than the wild-type counterpartto both paclitaxel and TNFa by almost 1.4-fold. In particular, thepercentage of HT-29PKC cells that survived after a 24 hr paclitaxeltreatment was about 85% (whereas in HT-29 culture a similartreatment led to a 58% survival): after 48 hr, TNFa treatmentsurvival was about 72% (whereas in HT-29 culture a similartreatment led to a 55% survival; Table II).Thus, it can be con-cluded that a high intracellular level of PKCb1 confers resistanceto apoptosis in HT-29 colon carcinoma cells. Taken together, theresults of the experiments reported in Figure 3 and Table II wouldimply that stimulation of conventional PKC protects HT-29 cellsmore from paclitaxel than from TNFa.

DISCUSSION

In the present work we assayed the sensitivity of HT-29 cells todifferent apoptogenic drugs, namely, etoposide (VP-16), TNFaand paclitaxel, which act through different cellular pathways; wewondered whether, together with p53 loss of function, altered PKCexpression would affect the response to these apoptotic treatments.Two main conclusions can be drawn from the present study: 1)TNFa and paclitaxel, but not etoposide, are cytotoxic for HT-29cells, presumably through p53-independent pathways; and 2)aandb PKCs are involved in the apoptotic pathways activated byTNFa and paclitaxel.

Etoposide at a concentration that was cytotoxic for many celltypes in vitro had a low cytotoxicity.13,15,16 This fact might be

FIGURE 3 – Phorbol myristate acetate (PMA)-induced stimulation ofPKC protects HT-29 cells from tumor necrosis factora (TNFa)- orpaclitaxel (Taxol[Tax])-induced apoptosis. Adherent HT-29 cells werecultured in serum-free medium and exposed or not to 50 nM PMA for24 hr or throughout the whole experimental period as indicated.Parallel cultures were treated for the last 48 hr with 100 ng/ml TNFa(a) or the last 24 hr with 50 nM paclitaxel (b). Medium was changeddaily and substances re-added. Cell survival in TNFa - or paclitaxel-treated cultures is calculated as percentage of their respective controls.Data represent the mean6 SD of three separate experiments.

FIGURE 4 – Expression of protein kinase Cb1 (PKCb1) protein inHT-29, HT-29C1 and HT-29PKC cells. The homogenates of HT-29,HT-29C1 and HT-29PKC cells were analyzed by western blotting forPKCb1 expression. (a) A representative gel. (b) The same filter wasstripped and re-probed with anti-b-actin antibodies to show equaltransfer of cellular proteins among samples. Specific bands on filmwere quantitated by densitometric analysis. (c) Data reported in thehistogram are normalized againstb-actin levels and represent theaverage6 SD of three determinations on three separate western blots.St, pre-stained standard molecular weight.

TABLE II – HYPER-EXPRESSION OF PKCbI AND SUSCEPTIBILITY OF HT-29CELLS TO TNFa- OR PACLITAXEL-INDUCED APOPTOSIS1

Cell line Co Paclitaxel TNFa

HT-29 100 586 4 556 4HT-29PKC 100 856 5 726 41Adherent HT-29 and HT-29PKC cultures were exposed to 100

ng/ml tumor necrosis factora (TNFa) for 48 hr or to 50 nM paclitaxelfor 24 hr as indicated. Cell survival calculated as in Table I. Datarepresent the mean6 SD of three independent experiments. Co,control.

182 CESAROET AL.

related to the mechanism of action of this drug that causes DNAdamage through inhibition of topoisomerase II.27 It is likely thatthe absence of functional p53 negatively influenced the onset ofapoptosis in etoposide-treated HT-29 cells. This interpretation isconsistent with the report by Loweet al.,1 who demonstrated thatp53-deficient fibroblasts developed resistance to apoptosis inducedby several DNA damaging agents, etoposide included. HT-29 cellshave been previously reported to be rather resistant to TNFa,which in this cell type mainly exerts cytostatic effects.28 In gen-eral, TNFa elicits apoptotic effects when administered along withmetabolic inhibitors such as cycloheximide or actinomycin D.Here we show that TNFa alone is cytotoxic for HT-29 cells in adose- and time-dependent manner. Cytotoxicity by this cytokinewas enhanced in HT-29 cells treated for as long as 48 hr inunchanged medium, a condition that negatively impacts on cellularmetabolism.

TNFa binds to its membrane receptor and triggers the activationof a cascade of kinases and proteases that culminate into apopto-sis.29 The extent of cytostatic or cytotoxic effects mediated byTNFa depends on the amount of specific receptors that are en-gaged as well as on the fine tuning of the intracellular signals thatare recruited upon their activation. Therefore, the cytokine con-centration, cell density and level of membrane expression of thereceptor as well as the integrity of the signaling pathways involvedare all factors that influence the outcome of the treatment. In thisrespect, it is worth noting that pre-incubation with interferongsensitized HT-29 cells to TNFa cytotoxicity.28,30 One can specu-late that interferong increases not only the synthesis31 but also thenumber of TNF receptors at the plasma membrane level. We foundthat co-treatment with an inhibitor of PKC (a andb isoforms) atdoses not cytotoxicper sefor as long as 48 hr sensitized HT-29cells to TNFa cytotoxicity. Conversely, PMA-induced stimulationof conventional PKC activity protected HT-29 cells from thiscytokine. We further assessed the role of theb isoform of PKC inthis process. To this end we employed a transfected clone ofHT-29 cells, which was shown to express about sevenfold higherlevels of the PKCb1 protein than the parental cell line (Fig. 4). Wefound that transfected HT-29PKC were more resistant to TNFa(and to paclitaxel) than their untransfected parental counterpart.

On the whole, the present data consistently demonstrate that PKCis clearly involved in TNFa cytotoxicity in HT-29 cells.

High rates of apoptosis were obtained by treating HT-29 cellswith paclitaxel, a chemotherapeutic drug not commonly used forthe treatment of colorectal cancer. Inhibition of PKC activityenhanced paclitaxel-induced cytotoxicity, and paclitaxel was con-sistently more effective on HT-29 cells than on HT-29PKC. More-over, paclitaxel was virtually not effective in cultures (pre-)treatedwith PMA under conditions that stimulateda andb PKC isoforms(Fig. 3). Therefore, PKC appears to be clearly involved in themolecular mechanisms initiated by paclitaxel and leading to apo-ptosis of colorectal cancer cells. Clinically, paclitaxel is an activeagent in the treatment of many tumor types including leukemia andbreast, ovary and prostate cancers.32–34 Based on data reportedhere, paclitaxel should be considered a potential candidate for thechemotherapeutic treatment of colon carcinomas and possibly alsoin association with inhibitors of PKC at doses not cytotoxicper se.

PKCb isoforms have been shown to be expressed at low levelsin colorectal cancer, and this has been linked to the loss of theenterocytic differentiated phenotype.7,9 Consistently with thisview, hyper-expression of PKCb1 was associated in HT-29 cellswith (partial) recovery of cell proliferation control and the abilityto acquire an enterocytic-like differentiated phenotype under ade-quate experimental conditions.11,26 Here we have shown that ac-tive PKCb1 (and probably other PMA-sensitive PKC isoforms)confers resistance in HT-29 cells exposed to various apoptogenicstimulus, apparently regardless of their type and mechanism ofaction. Therefore, altered expression of PKC should be consideredamong the mechanisms responsible for the resistance to cytotoxicdrugs exhibited by colorectal cancer cells.

ACKNOWLEDGEMENTS

Thanks are due to Dr. Weinstein (Columbia University, NewYork, NY) for providing us with the HT-29PKC and HT-29C1clones. R.C. was supported by a fellowship from the FondazioneCavalieri Ottolenghi (Torino, Italy). M.D. is a postdoctoral fellowat the Universita` del Piemonte Orientale “A. Avogadro”.

REFERENCES

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