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Cell Growth & Differentiation Vol. 11, 641-648, December 2000
© 2000 American Association for Cancer Research


Articles

Protein Kinase C ßI Is Implicated in the Regulation of Neuroblastoma Cell Growth and Proliferation1

Karin Svensson, Ruth Zeidman, Ulrika Trollér, Anna Schultz and Christer Larsson2

Lund University, Department of Laboratory Medicine, Molecular Medicine, Malmö University Hospital, 205 02 Malmö, Sweden

Abstract

To investigate a putative involvement of protein kinase C (PKC) isoforms in supporting neuroblastoma cell proliferation, SK-N-BE(2) neuroblastoma cells were transfected with expression vectors coding for the C2 and V5 regions from different PKC isoforms. These structures have been suggested to inhibit the activity of their corresponding PKC isoform. The PKC fragments were fused to enhanced green fluorescent protein to facilitate the detection of transfected cells. Expression of the C2 domain from a classical PKC isoform (PKC{alpha}), but not of C2 domains from novel PKC{delta} or PKC{epsilon}, suppressed the number of neuroblastoma cells positive for cyclin A and bromodeoxyuridine incorporation. This indicates a role for a classical isoform in regulating proliferation of these cells. Among the V5 fragments from PKC{alpha}, PKCßI, and PKCßII, the PKCßI V5 had the most suppressive effect on proliferation markers, and this fragment also displaced PKCßI from the nucleus. Furthermore, a PKCß-specific inhibitor, LY379196, suppressed the phorbol ester- and serum-supported growth of neuroblastoma cells. There was a marked enhancement by LY379196 of the growth-suppressive and/or cytotoxic effects of paclitaxel and vincristine. These results indicate that PKCßI has a positive effect on the growth and proliferation of neuroblastoma cells and demonstrate that inhibition of PKCß may be used to enhance the effect of microtubule-interacting anticancer agents on neuroblastoma cells.

Introduction

PKC3 comprises a family of at least 10 related serine/threonine protein kinases. Based on structural properties and cofactor requirements, the family is subgrouped into classical (PKC{alpha}, PKCßI, PKCßII, and PKC{gamma}), novel (PKC{delta}, PKC{epsilon}, PKC{eta}, and PKC{theta}), and atypical (PKC{iota} and PKC{zeta}) PKCs (1, 2, 3) . The enzymes of the PKC family have been implicated in the regulation of growth and differentiation in a number of cell types, and several studies suggest that different isoforms may have unique or even opposite effects on cell growth. For instance, in NIH3T3 cells, overexpression of PKC{epsilon} leads to an increased growth rate, whereas PKC{delta} has the opposite effects (4) , and in R6 embryo fibroblasts, PCKßI overexpression has a positive effect on cell growth, whereas PKC{alpha} has a negative effect on cell growth (5) . In vascular smooth muscle cells, the two closely related isoforms PKCßI and PKCßII have opposite effects, with PKCßI leading to a decreased doubling time and PKCßII leading to an increased doubling time (6) .

In SH-SY5Y neuroblastoma cells, treatment in the presence of serum with a phorbol ester concentration that does not down-regulate PKC leads to neuronal differentiation (7 , 8) . We have recently shown that PKC{epsilon} seems to be critical for the induction of neurites that accompanies neuronal differentiation (9) . On the other hand, for phorbol ester-mediated increased expression of differentiation marker genes such as neuropeptide Y, a classical PKC isoform is likely to be of importance (10) . PKC also seems to be able to support the growth of neuroblastoma cells. The growth of several neuroblastoma cell lines can be supported by phorbol ester in the absence of serum, and inclusion of PKC inhibitors in serum-containing medium leads to a suppressed growth of these cells (10) . The identity of the PKC isoform that mediates this effect is currently unknown.

One problem in determining which PKC isoform mediates a certain biological effect is the relative lack of isoform-specific inhibitors. However, in recent years, a picture has emerged suggesting that there may be isoform-specific docking proteins in the cell and that the interaction with these proteins may be essential for the activity of the isoform. RACKs constitute a group of proteins that bind activated PKC, and this interaction is mediated to a large extent by the C2 domain in the PKC molecule (11) . In line with these results, expression of isolated C2 domains has been shown to block the translocation and/or activation of PKC isoforms in a specific manner (12, 13, 14) .

The COOH-terminal V5 region, which comprises approximately 50 amino acids, has also been shown in several studies to mediate isoform-specific interactions or functions. This region is the only difference between the PKCßI and PKCßII isoforms and is thus likely to determine their unique functions. The V5 region from PKCßII was demonstrated to be the site for the specific interaction of this isoform with F-actin (15) and also to determine the specific function of PKCßII in erythroleukemia K256 cells (16) . The corresponding region was shown to be involved in the isoform-specific interaction of PKC{alpha} with the PDZ domain of PICK1 (17) and in the interaction of PKC with a PDZ domain of INAD (18) . The latter findings are particularly interesting because PDZ domain-containing proteins may act as scaffolds assembling components of a transduction cascade within one complex (19) . There are also results indicating a role for the V5 region in the binding of PKCßII to its RACK (20) .

The aim of this study was to elucidate which PKC isoform is important for neuroblastoma cell proliferation. This was done primarily by using an approach in which the above-mentioned domains of different PKC isoforms were introduced into the cells to inhibit endogenous PKC isoforms. The effects of this expression on proliferation markers were then analyzed.

Results

The C2 domain has been suggested to act as a PKC isoform-specific inhibitor when introduced into cells (11) . cDNAs coding for C2 domains from the PKC isoforms that are consistently expressed in neuroblastoma cells (PKC{alpha}, PKCß, PKC{delta}, and PKC{epsilon}) were amplified with PCR and introduced into the pEGFP-N1 expression vector, which generated a PKC C2-EGFP fusion protein when transfected into mammalian cells. PKC{alpha}, PKC{delta}, and PKC{epsilon} C2-EGFP fusions were readily expressed when introduced into SK-N-BE(2) cells (Fig. 1)Citation , whereas the corresponding fusion construct for PKCß generated essentially no fusion protein (data not shown).



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Fig. 1. PKC C2 and V5 domain constructs. This figure outlines the structure of classical (PKC{alpha}, PKCßI, and PKCßII) and novel (PKC{delta} and PKC{epsilon}) PKC isoforms and highlights in gray the C2 and V5 regions used in this study. SK-N-BE(2) cells were transfected with expression vectors coding for EGFP alone and for PKC{alpha}, PKC{delta}, and PKC{epsilon} C2 domains and PKC{alpha}, PKCßI, and PKCßII V5 domains fused to EGFP. Cell lysates (60 µg of cellular protein) were subjected to Western blot analysis with polyclonal anti-green fluorescent protein antibody. The positions of molecular weight markers (97, 66, 46, 30, and 21) are indicated (in thousands). Arrowheads indicate the positions of the V5 and C2 domain-EGFP fusion proteins. An unspecific band at approximately Mr 55,000 is also detected with this antibody.

 
The effect of expression of the PKC{alpha}, PKC{delta}, and PKC{epsilon} C2 constructs was evaluated for a possible influence on neuroblastoma cell proliferation using SK-N-BE(2) cells. Because the transfection efficiency of these cells is generally between 2% and 5% (9) , the expression of EGFP was used to identify transfected cells. After transfection, the cells were incubated with medium containing 10% FCS for 40 h, and then the percentage of transfected cells staining positive for two proliferation markers, incorporation of BrdUrd and cyclin A expression, was assessed (Fig. 2A)Citation . This experiment revealed that the C2 domain from PKC{alpha} suppressed the number of SK-N-BE(2) cells positive for the two proliferation parameters. The number of BrdUrd-positive cells was suppressed by 29%, and the number of cyclin A-positive cells was suppressed by 35% by expression of the PKC{alpha} C2 domain. These effects were statistically significant. The effects of C2 domains from PKC{delta} and PKC{epsilon} were minor (8–15% inhibition) and not significant, indicating that the inhibition is specific for the PKC{alpha} C2 domain. The fact that the results were similar for both BrdUrd incorporation and cyclin A staining indicates that either marker can be used to analyze proliferation of neuroblastoma cells.



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Fig. 2. The effect of PKC C2 domains on proliferation parameters of neuroblastoma cells. SK-N-BE(2) cells were transfected with expression vectors coding for C2 domains from the indicated PKC isoforms fused to EGFP and then cultured for 48 h in medium supplemented with 10% serum (A) or in the presence of 16 nM TPA in SHTE medium (B). BrdUrd incorporation ({blacksquare} in A and B) and detection of cyclin A with immunofluorescence ({square} in A) were used to estimate proliferating activity of transfected cells. Data are expressed as the percentage of transfected cells positive for either BrdUrd or cyclin A and are the mean ± SE of four (A) or three (B) separate experiments. Controls (-) are cells expressing EGFP only. Approximately 200 transfected cells identified by the presence of EGFP (A) or an average of around 100 transfected cells (B) were scored on each coverslip. Levels of significance: P < 0.05 (*) and P < 0.01 (**) compared with control cells (Student’s t test).

 
We have previously found that TPA can support the growth of neuroblastoma cells (10) . To investigate whether the PKC C2 domains have the same effects on TPA-treated neuroblastoma cells as they have on serum-supported cells, the incorporation of BrdUrd was analyzed after incubation with 16 nM TPA without serum after the transfection (Fig. 2B)Citation . As was the case for cells grown in the presence of serum, only the C2 domain of PKC{alpha} caused a potent statistically significant suppression (27%) of the number of cells positive for BrdUrd incorporation. Under these conditions, there were only minor nonsignificant effects of the PKC{delta} (16% inhibition) and PKC{epsilon} (6% inhibition) C2 domains.

The selective suppression of neuroblastoma cell proliferation markers by the C2 domain from PKC{alpha} suggests that this isoform may be of importance in regulating the proliferation of these cells. However, C2 domains of classical isoforms have a large degree of homology, and this, together with the fact that it was not possible to test the C2 domain of PKCß, precludes the drawing of definite conclusions.

We have previously demonstrated that mRNA for classical PKC isoforms PKC{alpha}, PKCßI, and PKCßII but not PKC{gamma} is present in SK-N-BE(2) neuroblastoma cells (10) . We also demonstrated the presence of PKC{alpha} and PKCßII protein, but due to antibody cross-reactivity, we could not determine whether PKCßI protein is present in neuroblastoma cells. To clarify this issue, several antibodies were examined for cross-reactivity with PKC{alpha} and PKCßII. To obtain extracts with large amounts of the different isoforms, expression vectors coding for PKC{alpha}, PKCßI, and PKCßII were constructed and transfected into CHO cells. This revealed that a monoclonal anti-PKCßI antibody from Santa Cruz Biotechnology displayed no cross-reactivity with PKC{alpha} or PKCßII and also showed that PKCßI protein is expressed in SK-N-BE(2) cells (Fig. 3, A and B)Citation . Hence, of the classical isoforms, these cells express PKC{alpha}, PKCßI, and PKCßII, and any of these may have been influenced by the PKC{alpha} C2 domain.



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Fig. 3. Expression of PKCßI and the effects of V5 domains of PKC{alpha} and PKCß isoforms on cyclin A expression in neuroblastoma cells. A, CHO cells were transfected with CMS-EGFP vectors containing cDNA coding for PKC{alpha}, PKCßI, or PKCßII. The different cell lysates were analyzed with Western blots and detected with isoform-specific antibodies that demonstrate that the PKC isoforms are generated from the expression vectors and that the antibodies are isoform specific. B, lysates of SK-N-BE(2) cells (BE) were analyzed with anti-PKCßI antibody. Lysates of CHO cells transfected with empty CMS-EGFP (-) vector or with vector containing cDNA for PKC{alpha}, PKCßI, or PKCßII were included as positive and negative controls. C, SK-N-BE(2) cells were transfected with expression vectors coding for V5 domains of PKC{alpha}, PKCßI, and PKCßII fused to EGFP. Two days after transfection, transfected cells were analyzed for cyclin A immunoreactivity. Data are expressed as the percentage of transfected cells positive for cyclin A and are the mean ± SE of three separate experiments. Approximately 200 transfected cells identified by the presence of EGFP were scored on each coverslip. Controls (-) are cells expressing EGFP only. Levels of significance: P < 0.01 (**) compared with control cells (Student’s t test).

 
The COOH-terminal V5 region of the PKC molecule is highly variable between the isoforms and is the only difference between PKCßI and PKCßII. This region is therefore a putative site for isoform-specific protein interactions, and expression of an isolated V5 region may perhaps block the corresponding endogenous isoform. Expression vectors coding for the V5 domain of the classical isoforms expressed in neuroblastoma cells (PKC{alpha}, PKCßI and PKCßII) fused to the COOH terminus of EGFP were constructed, and the generation of proteins of anticipated size was shown with Western blot after transfecting the constructs into SK-N-BE(2) cells (Fig. 1)Citation . The PKCV5-EGFP fusion proteins migrated as doublets, perhaps reflecting different states of phosphorylation of the two characterized phosphorylation sites in this region (21) . SK-N-BE(2) cells were transfected with the vectors, and the number of transfected cells expressing cyclin A was analyzed (Fig. 3C)Citation . The V5 region from PKCßI significantly suppressed the number of cyclin A-positive cells with 47%, whereas the V5 regions from PKC{alpha} and PKCßII had only minor effects on this parameter. This result highlights PKCßI as the PKC isoform of importance for neuroblastoma cell proliferation.

If the V5 domains act as inhibitors of PKC activity, it is likely that they localize to the same intracellular sites as the endogenous PKC isoform that they inhibit. The EGFP-PKCV5 fusion proteins all displayed a similar intracellular distribution (Fig. 4, A–C)Citation . There was a prominent localization to the nucleus of all fusion proteins, but in most cells, a presence in the cytosol could also be seen. Analysis of the intracellular distribution of the corresponding endogenous PKC isoforms (Fig. 4, D–F)Citation revealed that PKC{alpha} and PKCßII immunoreactivity was essentially localized to the cytosol, with no staining in the nucleus. In contrast, PKCßI was found primarily in the nucleus. To investigate whether the most effective V5 fragment, PKCßIV5, influences the nuclear localization of PKCßI, cells were transfected with expression vector coding for the EGFP-PKCßIV5 fusion protein. Endogenous PKCß was visualized with an antibody that reacts with the PKCß regulatory domain and thus detects both PKCß isoforms (Fig. 4, G–I)Citation . The staining with this antibody was generally weaker than that with the other PKCß antibodies, revealing PKCß immunoreactivity only in the nucleus and no reactivity corresponding to the PKCßII localization to the cytosol. In many cells overexpressing EGFP-PKCßIV5, there was a marked reduction in nuclear staining with the PKCß antibody. Because PKCßI is the isoform found in the nucleus, it suggests that the PKCßIV5 fragment indeed displaces PKCßI, implying that this isoform is inhibited by the PKCßIV5 fragment.



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Fig. 4. Intracellular distribution of overexpressed PKC{alpha}, PKCßI, and PKCßII V5 domains and corresponding endogenous PKC isoforms. A–C, SK-N-BE(2) cells were transfected with vectors coding for fusion proteins between EGFP and V5 domains from PKC{alpha} (A), PKCßI (B), and PKCßII (C). Images demonstrate the localization of the V5-EGFP proteins visualized by the fluorescence of EGFP. D–F, immunofluorescence analysis of endogenous PKC{alpha} (D), PKCßI (E), and PKCßII (F) in SK-N-BE(2) cells. The immunoreactivity was visualized with TRITC-conjugated secondary antibodies. G—I, SK-N-BE(2) cells were transfected with vector coding for the EGFP-PKCßI V5 fusion protein, and 16 h after transfection, the cells were analyzed with immunofluorescence using an anti-PKCß antibody, which is not directed against the V5 region, as primary antibody. The same microscopic field depicts (G) the EGFP-PKCßI V5 protein visualized by the fluorescence of EGFP, (H) immunoreactivity of the anti-PKCß antibody visualized by a secondary TRITC-conjugated antibody, and (I) a phase-contrast image.

 
The fact that expression of PKCßIV5 suppresses neuroblastoma cell proliferation raises the possibility that overexpression of PKCßI may augment proliferation of these cells. Expression vectors with cDNA for EGFP under the control of a SV40 promoter and PKCßI or PKCßII under the control of a cytomegalovirus promoter were transfected into SK-N-BE(2) cells. With these vectors, EGFP and PKC will be expressed as separate proteins, and the fluorescence of EGFP can be used to identify transfected cells. The generation of PKCßI and PKCßII from the respective plasmid was confirmed by Western blot analysis of CHO cells transfected with the vectors (Fig. 3A)Citation . The expression vectors coding for PKCßI and PKCßII were transfected into SK-N-BE(2) cells, and 40 h later, the incorporation of BrdUrd in transfected cells was assessed (Fig. 5)Citation . Overexpression of neither PKCß isoform had any marked effect on the percentage of cells staining positive for BrdUrd, which may indicate that the amount of PKCßI molecules is not rate-limiting for neuroblastoma cell proliferation.



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Fig. 5. The effect of overexpression of PKCßI and PKCßII on proliferation of neuroblastoma cells. SK-N-BE(2) cells were transfected with expression vector CMS-EGFP containing either no insert (-) or cDNA coding for full-length PKCßI (ßI) or PKCßII (ßII). The percentage of transfected cells positive for BrdUrd was counted. At least 200 transfected cells/experiment identified by the presence of EGFP were counted, and the data are mean ± SE.

 
Recently, inhibitors that are specific for the PKCß isoforms have been described (22) . To further establish that the PKCß isoforms are of importance for proliferation of neuroblastoma cells, the effect of such an inhibitor, LY379196, on the proliferation and growth was investigated. To quantify the proliferation under these circumstances, it was not necessary to analyze cell division activity at the single cell level, as it was with the transfection experiments. Therefore, incorporation of [3H]thymidine could be used as a measure of proliferation. SK-N-BE(2) cells that had been cultured in serum-free medium or medium supplemented with 16 nM TPA or 10% FCS were exposed to [3 H]thymidine, and after a 3-h incubation period, the incorporation was analyzed (Fig. 6A)Citation . The incorporation of [3 H]thymidine by cells that had been grown in serum-free medium or in medium supplemented with 16 nM TPA was suppressed by LY379196 in a concentration-dependent manner. Culture in the presence of 100 nM LY379196 led to a 28% suppression for cells grown in serum-free medium. There was a tendency toward a similar effect on TPA- and serum-supported cells, but the effect was not as pronounced. The concentration of LY379196 needed for this effect is in accordance with an effect on PKCß isoforms because the IC50 of this compound is 50 nM for PKCßI and 30 nM for PKCßII.4



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Fig. 6. Effect of LY379196 on proliferation and growth of neuroblastoma cells. The effect of the PKCß inhibitor LY379196 on the proliferation and growth of SK-N-BE(2) cells was investigated. A, SK-N-BE(2) cells were grown with increasing concentrations of LY379196 in SHTE medium ({square}), with 16 nM TPA () or with 10% serum ({blacksquare}). The cells were pulse-labeled with [3H]thymidine, and incorporated [3H]thymidine was recovered with cell harvesting. Data (mean ± SE; n = 9) are expressed as incorporated [3H]thymidine in percentage of values obtained in cells cultivated in the absence of LY379196. B, SK-N-BE(2) cells were grown with increasing concentrations of LY379196 for 3 days in the presence of 10% serum ({blacksquare}) or under serum-free conditions in the presence of 16 nM TPA (). The amount of viable cells were analyzed with a MTT assay. Data are expressed as a percentage of the values obtained in the absence of LY379196 and are the mean ± SE (n = 12). Levels of significance: P < 0.05 (*) and P < 0.01 (**) compared with no inhibitor (Student’s t test).

 
To investigate whether the suppressed proliferation rate by LY379196 results in a suppression of the growth of neuroblastoma cells, a MTT assay was used to estimate the amount of viable SK-N-BE(2) cells. This was measured after 3 days of culture in the presence of increasing concentrations of LY379196 (Fig. 6B)Citation . SK-N-BE(2) neuroblastoma cells were cultured in either regular growth medium (i.e., in the presence of 10% serum) or SHTE medium containing 16 nM TPA. For this parameter, there was a concentration-dependent effect of LY379196 for both culture conditions. Inclusion of 100 nM LY379196 in the medium caused a decrease in the amount of viable cells of 16% for cells cultured with serum and 30% for cells cultured with TPA.

By itself, the PKCß inhibitor did not completely block proliferation, and there was still a significant growth of neuroblastoma cells in the presence of LY379196. This raises the possibility that it is necessary to influence other mechanisms of importance for growth to detect a substantial effect of the PKCß inhibitor. LY379196 was therefore combined with increasing concentrations of the anticancer agents etoposide, paclitaxel, or vincristine. The effect of the drug combinations on the number of viable SK-N-BE(2) cells after culture for 3 days in regular growth medium was analyzed with a MTT assay. For etoposide, no substantial enhancement of the cytotoxic/growth-suppressive effect was observed when 100 nM LY379196 was included in the medium (data not shown). On the other hand, when paclitaxel or vincristine was combined with 100 nM LY379196, the potency of these drugs for reduction of viable cell number was enhanced (Fig. 7)Citation . For paclitaxel, the concentration that induced half maximal effect was reduced from 540 nM (range, 500–580 nM, two experiments) to 130 nM (range, 110–140 nM) in the presence of LY379196. The corresponding effect on vincristine was a reduction from 18 nM (range, 18–19 nM, two experiments) to 8 nM (range, 5–13 nM).



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Fig. 7. LY379196 potentiates the effect of paclitaxel and vincristine on the suppression of the number of viable neuroblastoma cells. SK-N-BE(2) cells were cultured for 3 days in regular growth medium with increasing concentrations of paclitaxel (A) or vincristine (B) in the absence ({circ}) or presence (•) of 100 nM LY379196. The viable cell number was analyzed with a MTT assay, and data (mean ± SE; n = 6) are expressed as a percentage of the values obtained in the absence of drugs.

 
Discussion

The aim of this study was to elucidate which PKC isoform is involved in regulating the proliferation of neuroblastoma cells and to investigate whether inhibition of this isoform could be used to attenuate the growth of these cells. To selectively block individual PKC isoforms, we used the fact that the introduction of RACK-interacting C2 domains has been shown to block activation and translocation of specific PKC isoforms (12, 13, 14) . C2 domains from different PKC isoforms were expressed in neuroblastoma cells, and the number of transfected cells positive for proliferation markers was estimated. This demonstrated that only a C2 domain from a classical isoform, PKC{alpha}, caused a substantial decrease in the amount of cells displaying markers representing proliferation activity. Significantly, the same pattern was observed for both BrdUrd incorporation and expression of cyclin A, clearly indicating that it is an effect on the amount of cells going through the cell cycle. It also demonstrates that either of these markers may be used to estimate the proliferation activity in these cells.

These results could suggest that PKC{alpha} has a positive effect on the proliferation of neuroblastoma cells. However, the C2 domains of PKC{alpha} and PKCß are highly homologous, and because the PKCß C2 construct could not be expressed, the effect of the PKC{alpha} C2 domain is not sufficient to conclude that PKC{alpha} is the isoform involved in proliferation. Neuroblastoma cells express PKC{alpha} and PKCßII (10) and PKCßI (Fig. 3B)Citation , and it is possible that any of these is the target for inhibition by the PKC{alpha} C2 domain.

In an attempt to distinguish the activities of these isoforms, we used the COOH-terminal V5 region, which comprises approximately 50 amino acids, and hypothesized that this fragment would function as an inhibitor of the respective isoform. The basis for the hypothesis is the fact that the V5 region is the only difference between the two PKCß isoforms and is thus likely to be involved in the specific effects of these isoforms. The V5 region has also been demonstrated to be involved in protein-protein interactions and PKC functions (15 , 17 , 18) . When the effects of the expression of V5 domains were analyzed in this study, it was evident that the V5 region from PKCßI, as opposed to V5 fragments from PKC{alpha} or PKCßII, caused a substantial suppression of the number of cyclin A-positive cells. This highlights PKCßI as a PKC isoform of relevance for neuroblastoma cell proliferation and adds to those studies that have suggested that introduction of the V5 fragment may be a fruitful approach for intervening with the function of certain isoforms.

However, it is not clear whether the V5 regions will function as isoform-specific inhibitors in all cellular settings. The isoform-selective effects of the C2 domains have been demonstrated by their capacity to specifically block the translocation of the corresponding isoform to membranes or other intracellular sites. The limited transfection efficiency of neuroblastoma cells only allows analysis of subcellular localization at the single cell level. The localization pattern of PKC{alpha} and PKCßII, which was rather diffuse in the cytosol, has not allowed us to determine whether the V5-EGFP fusion protein influences the localization or the activity of these isoforms. Despite the lack of effects of the PKC{alpha} and PKCßII V5 fragments on the proliferation parameters, the possibility that the activities of these isoforms are also of importance for neuroblastoma cell proliferation cannot be excluded. All PKCV5-EGFP fusion proteins displayed a high density in the nucleus, but a substantial amount of fluorescence could also be noted in the cytosol. Endogenous PKCßI was also shown to be present primarily in the nucleus, in contrast to PKC{alpha} and PKCßII, which were expressed in the cytosol. The PKCß immunoreactivity in the nucleus, most likely representing PKCßI, was frequently displaced in cells expressing PKCßIV5, which indicates that the effect of PKCßIV5 on proliferation is indeed due to interference with PKCßI function.

The hypothesis that a PKCß isoform is of importance for optimal proliferation activity of neuroblastoma cells is further supported by the effects of the PKCß inhibitor LY379196. Although the suppression of proliferation and growth by this compound was not as great as the effects of the PKCßIV5 region, the effect of LY379196 was obtained at concentrations that inhibit only the PKCß isoforms. The IC50 of LY379196 for PKCßI has been shown to be 50 nM, and the corresponding values for PKC{alpha}, PKC{delta}, and PKC{epsilon} are 600 nM, 700 nM, and 5 µM.4 Because concentrations higher than 100 nM may partially inhibit other PKC isoforms, higher concentrations were not used to exclude partial inhibition of other PKC isoforms. The maximal concentration used in our experiments (100 nM) was only two times the IC50 for PKCßI (50 nM) that would lead to a 60–70% inhibition of PKCßI, assuming Michaelis-Menten kinetics. The potentially remaining 30–40% PKCßI activity may explain the relatively smaller effect of LY379196 compared with the PKCßIV5 fragment.

The discrepancy between the two methods of inhibition could also be due to the fact that LY379196 will inhibit both PKCß isoforms, whereas the PKCßIV5 fragment may block only PKCßI. There are indeed reports indicating that PKCßI will augment and PKCßII will suppress proliferation in the same cell (6) . It is also possible that the BrdUrd assay with 30 min of labeling is more sensitive than the thymidine incorporation assay, in which there is a 3-h labeling period.

PKC has been shown to be involved in the regulation of proliferation in several cell systems, but the isoform of importance may vary depending on cell type. Overexpression of PKC{alpha} was shown to enhance smooth muscle cell proliferation (23) , whereas decreased PKC{alpha} levels led to increased proliferation of CaCo-2 cells (24) . Furthermore, overexpression of PKC{alpha} in 3Y1 cells was shown to suppress the activity of the transcription factor E2F that is positively linked to cell cycle progression (25) . A specific effect of PKCßI on proliferation and growth has been shown for rat fibroblasts (5) and vascular smooth muscle cells (6) . For both these cell types, overexpression of other classical isoforms, PKC{alpha} in fibroblasts and PKCßII in smooth muscle cells, resulted in a suppression of growth and proliferation. A PKCß isoform was shown to be positively involved in endothelial cell growth (26) , and PKCßII has been demonstrated to enhance the proliferation of colonic epithelium (27) . In fibroblasts, PKC{epsilon} was shown to enhance the growth rate, whereas PKC{delta} suppressed the growth rate (4) . Taken together, these data demonstrate that there are cell type-specific effects of different PKC isoforms on proliferation. This may reflect that PKC has no direct general effect on the cell division machinery but rather influences proliferation by modifying cell-specific pathways controlling the cell division. The present study highlights PKCßI as a PKC isoform with positive effects on neuroblastoma cell proliferation and growth.

When the PKCß inhibitor LY379196 was combined with paclitaxel or vincristine, a substantial enhancement of their effect was observed. This effect was obtained using 100 nM LY379196, a concentration specific for PKCß but not sufficient to obtain maximal inhibition of PKCßI. These data are in line with the finding that PKCß inhibition sensitized Lewis lung carcinoma cells to paclitaxel (28) and suggest that PKCß inhibition could also be used to increase the sensitivity of neuroblastoma cells to microtubule-interacting anticancer drugs.

In conclusion, this study provides indications that PKCßI is of importance for optimal neuroblastoma cell proliferation and growth. It suggests that introduction of a PKCßIV5 fragment into the cells is one way to intervene with this effect. It also shows that inhibition of a PKCß isoform, together with treatment with microtubule-interacting anticancer agents, may be a fruitful approach to attenuate the growth of neuroblastoma cells.

Materials and Methods

Plasmids.
Expression vectors coding for C2 domains of PKC{alpha}, PKCßI/ßII, PKC{delta}, and PKC{epsilon} and V5 domains from PKC{alpha}, PKCßI, and PKCßII fused to EGFP were generated by inserting cDNA coding for the C2 domains in the pEGFP-N1 vector (Clontech) and cDNA coding for the V5 domains in the pEGFP-C1 vector (Clontech). The cDNAs were generated with PCR using cDNA from SH-SY5Y cells (PKC{alpha}, PKCßII, PKC{delta}, and PKC{epsilon} constructs) and from IMR-32 neuroblastoma cells (PKCßI constructs) as template. Full-length cDNA for PKC{alpha}, PKCßI, and PKCßII was amplified and subcloned in the pEGFP-N1 vector, and then the NheI/SalI fragment containing full-length PKC cDNA was transferred to the CMS-EGFP vector (Clontech). The generation of PKC{alpha} cDNA has been described previously (9) . Restriction enzyme cleavage sites were introduced in the primers listed in Table 1Citation . The PCR reactions were done using Pfu polymerase, and the resulting cDNA products were sequenced to ensure that no mutations were introduced in the PCR reaction.


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Table 1 PKC C2 and V5 constructs used in this study

 
Cell Culture.
SK-N-BE(2) neuroblastoma cells were maintained in Eagle’s MEM supplemented with 10% FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. For transfection experiments, SK-N-BE(2) cells were trypsinized and seeded on coverslips at a density of 350,000 cells/35-mm dish. The following day, the cells were transfected using LipofectAMINE (Life Technologies, Inc.) according to the supplier’s protocol. For some experiments, cells were grown in SHTE medium [RPMI 1640 supplemented with 30 nM sodium selenite (Sigma), 10 nM hydrocortisone (ICN), 30 grams/ml transferrin (Roche), 10 nM ß-estradiol (ICN), 100 IU/ml penicillin, and 100 µg/ml streptomycin]. Cell culture solutions were obtained from Life Technologies, Inc.

Western Blot Analysis.
SK-N-BE(2) or CHO cells were transfected with different expression vectors, washed with PBS, and lysed in buffer [10 mM Tris (pH 7.2), 160 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EGTA, 1 mM EDTA, and Complete protease inhibitor mixture (Roche)]. Lysates were centrifuged for 10 min at 15,000 x g, and 20 µg (CHO cell lysates) or 60 µg [SK-N-BE(2) cell lysates] of protein were electrophoretically separated on a SDS polyacrylamide gel and then transferred to a Hybond-C extra nitrocellulose filter (Amersham). EGFP immunoreactivity was analyzed with antibodies directed against green fluorescent protein (Clontech) and detected with a horseradish peroxidase-labeled secondary antibody (Amersham) together with the SuperSignal system (Pierce). PKC{alpha} and PKCß isoforms were detected with isoform-specific antibodies (Santa Cruz Biotechnology). The chemiluminescence was detected with a charge-coupled device camera (Fujifilm).

BrdUrd Incorporation.
Cells were incubated with 10 µM BrdUrd for 30 min, fixed in 4% paraformaldehyde in PBS for 4 min, treated with 0.2 M HCl and 0.7% Triton X-100 in PBS for 10 min, washed several times with PBS, and incubated with 8 M urea for 30 min. Because the acid treatment abrogates the fluorescence of EGFP, both EGFP and BrdUrd were visualized with immunofluorescence. The cells were incubated for 30 min with primary antibodies [mouse anti-BrdUrd (Boehringer Mannheim; 1:10) and rabbit anti-GFP (Clontech; 1:200) in PBS], followed by a 30-min incubation with secondary antibodies, TRITC-conjugated donkey antimouse IgG and FITC-conjugated donkey antirabbit IgG (Jackson Laboratories) diluted 1:100 in PBS. The coverslips were mounted on microscope slides using a PVA-DABCO solution [9.6% polyvinyl alcohol, 24% glycerol, and 2.5% 1,4-diazabicyclo[2.2.2]octane in 67 mM Tris-HCl (pH 8.0)]. For experiments in Fig. 5Citation , cells were permeabilized in the absence of HCl and then incubated directly with the mouse anti-BrdUrd antibody together with DNase I (0.1 unit/µl) and exonuclease III (4 units/ml). Under these conditions, the fluorescence of EGFP could be used to identify transfected cells. Using fluorescence microscopy, the percentage of EGFP-expressing cells that were BrdUrd positive was counted.

Immunofluorescence.
Cells were fixed with 4% paraformaldehyde in PBS for 4 min and treated for 30 min with 5% donkey serum and 0.3% Triton X-100 in TBS. The cells were then incubated for 60 min with primary antibody rabbit anti-cyclin A diluted 1:150 in TBS, rabbit anti-PKC{alpha} (1:400), -PKCßI (1:400), -PKCßII (1:100; all from Santa Cruz Biotechnology), or with mouse anti-PKCß (1:50; Transduction Laboratories). After extensive washing, cells were incubated with TRITC-conjugated donkey antirabbit or antimouse IgG (Jackson Laboratories) for 60 min. The coverslips were mounted, and for cyclin A experiments, transfected cells were scored for cyclin A positivity.

[3H]Thymidine Incorporation.
SK-N-BE(2) cells were seeded at a density of 8000 cells/well in 96-well culture plates. The indicated compounds, TPA (Sigma) and LY379196 (kindly provided by Dr. D. K. Ways, Eli Lilly, Indianapolis, IN), had been added in 50 µl of medium before the addition of cells in the same volume. Two days after seeding, 20 µCi of [3 H]thymidine were added to each well, and after incubation for 3 h, incorporated [3 H]thymidine was recovered with a cell harvester (Skatron). The radioactivity was measured with scintillation counting.

Analysis of Cell Growth/Viability.
SK-N-BE(2) neuroblastoma cells were seeded at a density of 3000 cells/well in 96-well culture plates. The indicated compounds had been added in 50 µl of medium before the addition of cells in the same volume. After 3 days, the amount of viable cells was analyzed measuring the conversion of the tetrazolium salt MTT to formazan according to the supplier’s protocol (Promega). Paclitaxel (Sigma) was solubilized in DMSO, and vincristine sulfate (Sigma) was solubilized in water. For paclitaxel, control cells were incubated with 0.4% DMSO. For calculations of the concentration that elicited a half-maximal effect, a nonlinear curve fit was performed with the equation y = A2 + (A1- A2)/(1 + x/x0) where x0 corresponds to the half-maximal concentration.

Acknowledgments

Dr. D. K. Ways is gratefully acknowledged for providing LY379196 and for fruitful suggestions and comments.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Swedish Cancer Society; the Swedish Medical Research Council; the Child Cancer Foundation of Sweden; the Swedish Society for Medical Research; the Crafoord Foundation; Magnus Bergvall Foundation; Gunnar, Arvid, and Elisabeth Nilsson Foundation; Ollie and Elof Ericsson Foundation; John and Augusta Persson Foundation; and Malmö University Hospital Research Funds. Back

2 To whom requests for reprints should be addressed, at Lund University, Molecular Medicine, Entrance 78, 3rd Floor, Malmö University Hospital, 205 02 Malmö, Sweden. Phone: 46-40-337404; Fax: 46-40-337322; E-mail: Christer.Larsson{at}molmed.mas.lu.se Back

3 The abbreviations used are: PKC, protein kinase C; EGFP, enhanced green fluorescent protein; RACK, receptor for activated C-kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; BrdUrd, bromodeoxyuridine; CHO, Chinese hamster ovary; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TRITC, tetramethylrhodamine isothiocyanate. Back

4 D. K. Ways, personal communication. Back

Received for publication 5/10/00. Revision received 9/ 6/00. Accepted for publication 9/27/00.

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