The long-term accumulation of genetic aberrations plays a major role in the development of various cancers. Molecular abnormalities include expression of oncogenes, such as Ki-ras, c-myc, c-erb-B-2 and bcl-2, and tumour suppressor genes, such as p53, Rb and FHIT (fragile histidine triad) in lung cancer (Otterson et al, 1998; Salgia and Skarin, 1998).
We previously demonstrated that expression of an actin-regulatory protein (Yin and Stossel, 1979), gelsolin, is frequently downregulated in lung cancer (Dosaka-Akita et al, 1998) and several types of different human cancers, such as stomach, bladder and colon (Moriya et al, 1994; Tanaka et al, 1995; Furuuchi et al, 1996). Moreover, we indicated that introducing human cytoplasmic gelsolin cDNA suppresses tumorigenicity in human bladder and colon cancer cell lines (Tanaka et al, 1995; Furuuchi et al, 1996). Gelsolin controls the length of actin polymers in vitro by a variety of mechanisms. At least three different activities are known: severing, capping and nucleating through interaction with both filamentous (F-) and monomeric (G-) actins are responsible for reorganisation of the actin cytoskeleton. These functions are tightly regulated by calcium ions (Ca2+), pH and polyphosphoinositides (Janmey, 1994). Although gelsolin may modulate phospholipid signaling pathways through its high affinity to polyphosphoinositides (Janmey and Stossel, 1987), the mechanism that restored gelsolin expression suppresses tumorigenicity in cancer cells is not well understood.
Protein kinase C (PKC) belongs to a ubiquitous family of serine/threonine kinases that plays a critical role in many signal transduction pathways (Nishizuka, 1984). PKCs are activated by a variety of extracellular stimuli that elicit production of a lipid second messenger diacylglycerol (DAG) and a cofactor phosphatidylserine (PS) (Kishimoto et al, 1980; Nishizuka, 1992; Toker, 1998). Up to 12 different PKC isoforms have been reported in mammalian cells (Dekker and Parker, 1994; Mellor and Parker, 1998). The isoforms have been divided into three distinct subfamilies: the conventional PKCs (cPKCα, βI, II and γ), which are activated by DAG, PS and Ca2+; novel PKCs (nPKCδ, , η, θ and μ), which are dependent on DAG and PS, but not on Ca2+; and atypical PKCs (aPKCζ, ι/λ), which are activated by PS, but not by DAG and Ca2+ (Toker et al, 1994). Several studies have indicated that the individual PKC isoforms express in their tissue specifically and vary in biochemical properties and intracellular localisation (Nishizuka, 1992).
PKCs are shown to be a target for phorbol ester, such as 12-O-tetradecanoylphorbol-13-acetete (TPA) which is known as a tumour promoter, because TPA contains a diacylglycerol-like structure (Castagna et al, 1982; Gopalakrishna and Barsky, 1988). Several studies showed that overexpression of different PKC isoforms induces high growth rates, cellular saturation densities and enhanced tumorigenicity (Housey et al, 1988; Persons et al, 1988; Cacace et al, 1993; Mischak et al, 1993; Borner et al, 1995). Furthermore, the transfection of antisense PKCα into lung cancer cells repressed cell proliferation and reduced tumorigenicity in nude mice (Wang et al, 1999). However, there are a number of conflicting reports that overexpression of PKC increases or suppresses tumorigenicity in various cells (Janik et al, 1994; Johnson et al, 1999). These observations suggest that various PKCs play various roles in carcinomas and distinctive roles in cell regulation.
In this study, we established lung cancer cells (squamous cell carcinoma, PC10) overexpressing human cytoplasmic gelsolin in various degrees by gene transfer, using retrovirus carrying human cytoplasmic gelsolin cDNA, and examined the effects of restoration of gelsolin expression on tumorigenicity in vivo with nude mice and growth properties in culture in vitro. Moreover, we investigated bradykinin-induced responses, particularly inositoltriphosphate (IP3) production and translocation of various PKC isoforms from the cytosolic fraction to the particulate fraction in gelsolin-overexpressing clones, because agonist-stimulated activation of the phospholipases C (PLC)/PKC signal transduction pathway is critical for cell proliferation and tumorigenicity.