Report |
Address correspondence to Alan P. Fields, Mayo Clinic Comprehensive Cancer Center, Griffin Cancer Research Building, 4500 San Pablo Rd., Jacksonville, FL 32224. Tel.: (904) 953-6109. Fax: (904) 953-0277. email: fields.alan{at}mayo.edu
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Abstract |
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Key Words: Rac1; transgenic mice; rat intestinal epithelial cells; cell invasion; soft agar growth
Abbreviations used in this paper: ACF, aberrant crypt foci; AOM, azoxymethane; caPKC
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Introduction |
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Results and discussion |
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Given the relationship between PKC and Ras signaling (Uberall et al., 1999; Coghlan et al., 2000; Kampfer et al., 2001), we assessed whether PKC
is important for Ras-mediated transformation of the intestinal epithelium. We and others (Sheng et al., 2000; Murray et al., 2002; Yu et al., 2003) have used rat intestinal epithelial (RIE) cells to study Ras-mediated transformation, and elucidate the molecular mechanisms by which PKCßII promotes carcinogenesis. Ras-transformed RIE (RIE/Ras) cells were transfected with FLAG-tagged, wild-type (wt) PKC
or kdPKC
. Both RIE/Ras/wtPKC
and RIE/Ras/kdPKC
cells expressed elevated levels of PKC
when compared with RIE or RIE/Ras cells (Fig. 4 a, top). Immunoblot analysis using an antibody to oncogenic V12 Ras demonstrated that RIE/Ras, RIE/Ras/wtPKC
, and RIE/Ras/kdPKC
cells express comparable levels of oncogenic Ras (Fig. 4 a, second from top). Actin immunoblots confirmed that equal amounts of protein were loaded for each cell line (Fig. 4 a, third from top).
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RIE/Ras cells exhibited an increase in anchorage-dependent growth rate and saturation density compared with RIE cells (Fig. 4 b). Expression of wtPKC or kdPKC
had little effect on the Ras-mediated increase in growth rate or saturation density (Fig. 4 b). RIE cells expressing wtPKC
or kdPKC
in the absence of oncogenic Ras exhibited no demonstrable change in growth rate compared with RIE cells, and no signs of cellular transformation (unpublished data).
Because Ras transformation is dependent on activation of the small molecular weight GTPase, Rac1 (Qiu et al., 1995), we measured Rac1 activity in RIE/Ras cells (Fig. 4 c). As expected, RIE/Ras cells exhibit elevated Rac1 activity when compared with RIE cells (Fig. 4 c). Expression of either a dominant negative Rac1 (RacN17) mutant (Qiu et al., 1995) or kdPKC blocked Ras-mediated Rac1 activation. In contrast, expression of a constitutively active Rac1 (RacV12) mutant (Qiu et al., 1995) had little effect on Ras-mediated activation of endogenous Rac1. Expression of wtPKC
in the absence of oncogenic Ras was not sufficient to induce Rac1 activity (unpublished data). Thus, oncogenic Ras activates Rac1 in a PKC
-dependent fashion.
Both Ras and Rac1 have been implicated in cellular motility and invasion (De Corte et al., 2002) and RIE/Ras cells exhibit an invasive phenotype (Fujimoto et al., 2001). Therefore, we assessed whether the invasive phenotype observed in RIE/Ras cells is dependent on Rac1 and PKC. As expected, RIE/Ras cells are highly invasive, whereas RIE cells are not (Fig. 4 d). Expression of RacN17 or kdPKC
in RIE/Ras cells blocks Ras-mediated invasion (Fig. 4 d). However, expression of RacV12 in RIE/Ras/kdPKC
cells partially restores invasiveness. Thus, oncogenic Ras-mediated cellular invasion is dependent on both Rac1 and PKC
. Interestingly, expression of either wtPKC
or caPKC
in the absence of oncogenic Ras failed to induce invasion, indicating that PKC
is necessary for Ras-mediated invasion, but is not sufficient to induce invasion in the absence of oncogenic Ras (unpublished data).
RIE/Ras cells exhibit anchorage-independent growth in soft agar, whereas RIE cells do not (Fig. 5, a and b). Expression of wtPKC significantly enhances, and expression of kdPKC
blocks, soft agar growth of RIE/Ras cells (Fig. 5, a and b). Furthermore, expression of RacV12 in RIE/Ras/kdPKC
cells restores soft agar growth (Fig. 5 c). Expression of RacV12 in RIE cells in the absence of oncogenic Ras does not induce soft agar growth, indicating that expression of active Rac1 alone is not sufficient to cause cellular transformation (Fig. 5 c), which is consistent with previous reports that RacV12 exhibits very weak transforming potential (Khosravi-Far et al., 1995). These data demonstrate that PKC
plays a critical role in Ras-mediated transformation of RIE cells because PKC
is required for Ras-mediated activation of Rac1, cellular invasion, and anchorage-independent growth. Our data place PKC
downstream of oncogenic Ras and upstream of Rac1 in a pathway that stimulates invasiveness and soft agar growth, two hallmarks of the transformed phenotype. Next, we assessed the importance of PKC
in Ras-mediated colon carcinogenesis in vivo using transgenic mice expressing a latent oncogenic K-ras allele (G12D) that is activated by spontaneous recombination (Johnson et al., 2001). Latent K-ras (K-RasLA2) mice develop Ras-dependent lung carcinomas and colonic ACF (Johnson et al., 2001). We bred our transgenic kdPKC
mice with K-RasLA2 mice to generate bitransgenic K-RasLA2/kdPKC
mice, and assessed them for spontaneous ACF development (Fig. 5 d). K-RasLA2/kdPKC
mice developed significantly fewer ACF in the proximal colon than K-RasLA2 mice. These data are consistent with our results in RIE/Ras cells in vitro, and demonstrate that PKC
is critical for oncogenic K-rasmediated colon carcinogenesis in vivo.
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In this report, we present conclusive evidence that PKC is critical for colonic epithelial cell transformation both in vitro and in vivo. Interestingly, disruption of PKC
signaling by kdPKC
has little effect on normal intestinal epithelial cell homeostasis in vitro and in vivo, suggesting that PKC
may be an attractive target for development of novel therapeutics against colon cancer.
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Materials and methods |
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Production of transgenic mice and carcinogenesis studies
Human caPKC and kdPKC
cDNAs were generated and characterized previously (Jamieson et al., 1999; Lu et al., 2001). Transgenic caPKC
and kdPKC
mice were generated on a C57Bl/6 background using the Fabpl4x at -132 promoter (Simon et al., 1997; provided by J. Gordon, Washington University, St. Louis, MO) to direct transgene expression to the colonic epithelium (Murray et al., 1999). Isolation of colonic epithelium, immunoblot analysis for PKC
, and immunoprecipitation histone kinase assays were described previously (Jamieson et al., 1999; Murray et al., 1999). Transgenic caPKC
, transgenic kdPKC
, and nontransgenic mice were injected with either 10 mg/kg AOM or saline as described previously (Gökmen-Polar et al., 2001). ACF analysis was performed 12 wk after the last AOM injection (Murray et al., 2002) using well-defined criteria (McLellan et al., 1991). Mice were analyzed at 40 wk for tumor number, size, location, and pathological grade as described previously (Gökmen-Polar et al., 2001). All tumors were classified as either tubular adenomas or intramucosal carcinomas (carcinoma in situ) by Z. Gatalica, a board-certified pathologist. Digital images of the tumors were captured on a microscope (model Eclipse E600; Nikon) equipped with a ProgRes C14 camera (Jenoptik) using a 20x objective lens. Images were acquired using ProgRes C14 software with Microsoft Photoeditor and processed with Microsoft Photoshop.
Transgenic K-rasLA2 mice (Johnson et al., 2001; provided by T. Jacks, Massachusetts Institute of Technology, Cambridge, MA) were bred to transgenic kdPKC mice to obtain bitransgenic K-rasLA2/kdPKC
mice. At 12 wk old, transgenic K-rasLA2 and bitransgenic K-rasLA2/kdPKC
mice were assessed for spontaneous ACF formation (McLellan et al., 1991; Murray et al., 1999).
RIE cell transfections and cellular analyses
RIE cells and derivatives were grown in DME containing 5% FBS as described previously (Ko et al., 1998). RIE/Ras cells were described elsewhere (Sheng et al., 2000; provided by H.M. Sheng, University of Texas Medical Branch [UTMB], Galveston, TX). Microarray analysis of RIE/Ras cells demonstrated that these cells do not express PKC (unpublished data). Human wtPKC
and kdPKC
cDNAs were cloned into the pBABE/FLAG/puro retroviral expression vector and virus stocks were produced using Phoenix-E cells (provided by G. Nolan, Stanford University, Palo Alto, CA). Puromycin-resistant, stable transfectants were generated as described at http://www.stanford.edu/group/nolan/retroviral_systems/retsys.html. Expression of FLAG-epitopetagged PKC
was confirmed by immunoblot analysis using anti-FLAG antibody (Sigma-Aldrich), and PKC
kinase activity was determined by immunoprecipitation histone kinase assay as described previously (Jamieson et al., 1999).
Recombinant retroviruses containing Myc-tagged RacN17 or Myc-tagged RacV12 were generated by excising the Myc-tagged Rac1 constructs from pEXV/Rac vectors (Qiu et al., 1995) with EcoRI and ligating them into the EcoRI site of the LZRS-GFP retrovirus. The entire coding sequence of each construct was confirmed by DNA sequence analysis. LZRS-GFP-Rac1 retroviruses were used to infect RIE cells and derivative cell lines using a protocol described previously (Ireton et al., 2002). Rac1 activity was assessed by affinity isolation of GTP-bound Rac1 using a protocol described previously (Sander et al., 1998). Active GTP-bound Rac1 and total Rac1 were identified by immunoblot analysis using a Rac1 mAb (BD Biosciences) and quantitated by densitometry.
Invasiveness of RIE cell transfectants was assessed in Transwell inserts precoated with Matrigel (6.5-mm diam, 8-µm pore size; BD Biosciences). DME containing 10% FBS was added to the bottom chamber and 5 x 104 cells were suspended in 500 µl of serum-free DME and placed in the top chamber of the Transwell insert. Cells were incubated for 22 h at 37°C in 5% CO2, at which time noninvading cells were removed from the top chamber. Cells that had invaded through the Matrigel-coated filter were fixed in 100% methanol, stained with crystal violet, and counted on a microscope (Nikon) using a calibrated ocular grid. 15 representative areas of the bottom chamber were counted to determine the number of invasive cells in each well.
To assess anchorage-independent growth, RIE cell transfectants were suspended in DME supplemented with 10% FBS, 1.5% agarose, and a 1% insulin, transferrin, and selenium solution (Sigma-Aldrich), and plated (300 cells/60-mm dish) on a layer of 1.5% agar containing the same medium. Cell colonies were fixed with 20% methanol and stained with Giemsa after 714 d in culture and quantified under a dissecting microscope (Nikon).
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health to A.P. Fields (CA81436) and N.R. Murray (CA94122), and by an Institutional American Cancer Society grant (N.R. Murray). This work was initiated while the authors were at UTMB.
Submitted: 4 November 2003
Accepted: 22 January 2004
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