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Summary: Cytokeratins (CKs) constitute the largest family of intermediate filament proteins, and are subdivided into type I (CK9–CK23) and type II (CK1–CK8) subclasses. CK19 is expressed in non-small cell lung cancer (NSCLC), and serum CK19 fragment (referred to as CYFRA21-1) is one of the tumor markers used in diagnosing NSCLC. Type I and type II CKs have been shown to form obligate 1:1 heteropolymers, suggesting that dynamic changes must occur in the expression levels of CK pools when one CK is suppressed. However, the absolute levels of CK expression and their dynamic changes have not been fully evaluated. Therefore, we quantitatively determined CK expression levels in NSCLC cell lines, and evaluated the rate of change of CK expression levels after RNA interference targeting of single CKs. In NSCLC cells, type I CK18 and type II CK8 are the dominant CKs, with absolute expression levels of 12–77pmol/106 cells, while the expression patterns of the CKs vary among cell lines. Moderate suppression of a single dominant CK caused downregulation in CKs of the complementary type, and upregulation of other CKs of the same type. In contrast, severe suppression of a single dominant CK caused almost complete suppression of all CKs. In addition, introduction of CK19 led to resistance to CK degradation by CK18 suppression. These data suggest the presence of a critical threshold expression level for a dominant CK and a role for CK19 in the compensation of type I and type II CK pools in NSCLC. [Copyright &y& Elsevier]

Carbonic anhydrase-related protein VIII (CA-RP VIII) is expressed in most non-small cell lung cancer, and especially strongly at the front of tumor progression. Screening analysis of CA-RP VIII expression in a panel of cultured lung cancer cell lines showed that a well differentiated adenocarcinoma cell line, PC-9, appeared to lack CA-RP VIII. Subsequently, CA8 cDNA was transfected with an expression vector into PC-9. Ectopic overexpression of CA-RP VIII reduced the growth of PC-9 cells on uncoated culture dishes, especially when the cultures were started at low cell density, but increased cell growth on laminin-coated dishes. Interestingly, ectopic CA-RP VIII expression markedly reduced caspase-3 activity induced by serum starvation and anti-cancer agents in PC-9 cells. The present findings suggest that CA-RP VIII expression promotes progression of lung cancer by multifarious mechanisms. [Copyright &y& Elsevier]

Cytokeratin 8 (CK8) is one of the cytoskeletal components and shows caspase-mediated degradation when cells undergo apoptosis. We previously reported that CK8 is highly expressed in non-small cell lung cancer (NSCLC) cell lines and increasing values of serum CK8 are significantly associated with tumor progression in patients with NSCLC. In this investigation, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in lung cancer cell lines, revealed a shorter PCR product, which differed from the wild-type product of CK8. The nucleotide sequence of the shorter PCR products and genomic DNA for CK8 demonstrated that the shorter product was an aberrantly spliced form of CK8 (AS-CK8) which lacked a caspases cleavage site within the linker lesion in exon 5. The putative protein products predicted by the mRNA of AS-CK8 were demonstrated by Western blotting with monoclonal antibodies for CK8. In addition, AS-CK8 mRNA and its protein products were highly expressed in NSCLC cell lines compared with small-cell lung cancer (SCLC) cell lines. Tissue samples obtained from NSCLC patients also expressed mRNA of AS-CK8. In conclusion, we identified aberrantly spliced CK8 (AS-CK8) which lacked a caspases cleavage site in lung cancer cell lines and primary tumors of NSCLC. AS-CK8 was preferentially expressed in NSCLC, rather than SCLC. These findings lead to speculation that cancer cells expressing AS-CK8 may have a resistance to apoptosis and may perturb keratin network formation. [Copyright &y& Elsevier]

It has been reported that cytokeratin 8 (CK8) can be expressed in several cancers and expression of CK8 is correlated with increased invasiveness of the tumor in vitro and in vivo. In the present study, we investigated expressions of CK8 in human lung cancer cell lines. In addition, we also evaluated the clinical significance of CK8 measurements in sera of patients with lung cancer. Expression of mRNA for CK8 was semi-quantitatively evaluated by the competitive reverse transcriptase-polymerase chain reaction (competitive RT-PCR), using human lung cancer cell lines. The level of CK8 protein in culture supernatants of lung cancer cell lines and 70 sera of patients with lung cancer was measured by enzyme-linked immunosorbent assay (ELISA). Levels of serum CK8 according to clinical parameters were also examined. The level of expression of CK8 mRNA in non-small cell lung cancer (NSCLC) cell lines was significantly high compared with that of small cell lung cancer (SCLC) cell lines (P<0.05). The level of CK8 in culture supernatants in NSCLC was significantly high compared with that of SCLC. The level of serum CK8 in patients with NSCLC was significantly high compared with that of normal non-smokers and compared with that of SCLC (P<0.05). Patients with a CK8 value of 50.0 ng/ml, or higher, had a statistically significant diminished survival compared with those patients whose CK8 values were lower. In conclusion, CK8 was preferentially expressed in NSCLC. Increasing values of CK8 were significantly associated with tumor progression and decreased survival in patients with NSCLC. Therefore, CK8 in sera may become a novel tumor marker in patients with lung cancer. [Copyright &y& Elsevier]