-Phase kinase associated protein 2 (SKP2), a member of the F-box family, is the substrate-recognition subunit of the SCF SKP2 ubiquitin ligase complex. 1) SKP2 has been implicated in ubiquitin-mediated degradation of the cyclin-dependent kinase (CDK) inhibitor p27 KIP1 , and positively regulates the G1/S transition. [2][3][4] This molecule is also required for ubiquitination of other cell-cycle regulators including free cyclin E (non-CDK2 bound), 5) E2F1, 6) and hOrc1p. 7) SKP2 knock-out mice grow more slowly and have smaller organs than littermate controls 5) ; cells in these mutant mice contain enlarged nuclei with polyploidy and multiple centrosomes, and they show a reduced growth rate and increased apoptosis.Several lines of evidence have suggested an oncogenic role for SKP2. For example, double-transgenic mice expressing both activated N-Ras and SKP2, targeted to the T-lymphoid lineage, developed T cell lymphoma.8) Over-expression of SKP2 has been observed in transformed cells 2) and in various types of human tumors. [8][9][10][11][12][13] Expression of SKP2 correlates with the grade of malignancy in lymphomas 8) and oral squamous-cell carcinomas.11) Elevated expression of SKP2 indicates poor prognoses for patients with oral squamous-cell carcinomas 12) and gastric cancers.13) However, little is known about the mechanism of SKP2 over-expression in cancer cells or the nature of its contribution to the malignant phenotype.Earlier studies in our laboratory revealed that the 5p11-p13 region was frequently amplified in cell lines derived from small-cell lung carcinomas (SCLCs).14) We identified a probable target gene within this amplicon, SKP2, whose amplification was associated with tumorigenesis; SKP2 was amplified in 44% and over-expressed in 83% of the primary SCLC tumors we examined. Expression of SKP2 was inversely correlated with expression of p27 KIP1 , and down-regulation of the former by transfection of an antisense oligonucleotide inhibited the growth of SCLC cells in culture. Those results indicate that amplification of chromosomal DNA is one of the mechanisms capable of up-regulating SKP2, and that consequent overexpression of this gene plays an important role in the growth of SCLC cells.In the study reported here, we explored the mechanism(s) of the observed growth inhibition induced by down-regulation of SKP2. We transfected antisense oligonucleotides into three lung-cancer cell lines that had exhibited amplification and consequent over-expression of the gene, one line derived from an SCLC and two lines from non-small cell lung cancers (NSCLCs). Antisense treatment inhibited growth of all three populations. Because SKP2 promotes progression to Sphase, 2, 4) we first tested the cultures for reduction of DNA synthesis and then examined whether apoptotic processes were contributing to the decrease in growth. Our results provide the first evidence that reducing SKP2 with antisense nucleotides can directly induce apoptosis in lung-cancer cells.

A human non-small-cell lung carcinoma cell line, Calu-6 (from an anaplastic carcinoma), was transfected with the Ki-ras-related anti-oncogene Krev-1. Several transfectant lines were obtained that showed a reduced tumorigenicity in nude mice with respect to the parental and control transfected cell lines. This decrease was approximately 50% in tumor incidence at 4 wk after subcutaneous inoculation of the transfected cells. In addition, the volume of the Calu-6 revertant-derived tumors was three to 10 times smaller than that of the equivalent tumors produced by inoculation of the control cell line transfected with the neomycin-resistance gene. Krev-1--transfected cells that exhibited reduced tumorigenicity expressed Krev-1 mRNA and had variable numbers of copies of the Krev-1 gene. Moreover, Krev-1--transfected cells exhibited a more differentiated squamous epithelial morphology than the parental and control cell lines did. Moderately elevated levels of protein kinase C activity were detected in some revertant clones. Such activity correlated with the level of expression of Krev-1 mRNA in most cases. In summary, Krev-1 induced important morphological and biological changes in transfected Calu-6 cells that we interpreted as partial reversion of the malignant phenotype.

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