Sarcoendoplasmic reticulum Ca²⁺ ATPase type 2 downregulated in human oral squamous cell carcinoma

Abstract

Mice with a heterozygous deletion of the Atp2a2 gene (Atp2a2 ؉/-) encoding SERCA2 spontaneously develop SCCs of the skin and upper digestive tract, including the oral cavity. To elucidate the contribution of ATP2A2 to human oral carcinogenesis, we analyzed genetic and epigenetic changes as well as mRNA and protein expression in primary OSCCs and OPLs. With the exception of one OSCC-derived cell line showing a 12 bp deletion of ATP2A2, we found no mutations in the coding sequence of the gene in primary OSCCs (n ‫؍‬ 52), OPLs (n ‫؍‬ 32) and cell lines (n ‫؍‬ 8). In immunohistochemistry, however, high frequencies of ATP2A2 downregulation were evident not only in primary OSCCs (42%, 42/100) but also in OPLs (31%, 10/32). Real-time quantitative RT-PCR data were consistent with the protein expression status. Aberrant DNA methylation within ATP2A2 also was detected in 9 of 30 ATP2A2-downregulated OSCCs. Moreover, restoration or elevated expression of the ATP2A2 protein was induced in most of the cell lines showing ATP2A2 methylation after treatment with 5-aza-2 -dC, a DNA demethylating agent. These results suggest that inactivation of the ATP2A2 gene is a frequent and early event during oral carcinogenesis and that loss of expression may be regulated partly by an epigenetic mechanism.

Overexpression of stathmin in oral squamous-cell carcinoma: correlation with tumour progression and poor prognosis

“…PVDF membranes were washed again and incubated with a 1 : 1000 of horseradish peroxidase-conjugated antirabbit IgG Envision þ (Dako Japan Inc., Kyoto, Japan) as a secondary antibody for 20 min at room temperature. Finally, the membranes were incubated with ECL þ -horseradish peroxidase substrate solution included in the ECL þ kit (Amersham Biosciences UK Ltd, UK), and immunoblotting was visualised by exposing the membrane to Hyperfilm (Amersham) (Endo et al, 2004;Kasamatsu et al, 2005).…”

Ubiquitous mitochondrial creatine kinase downregulated in oral squamous cell carcinoma

“…All cell lines were maintained at 371C (humidified atmosphere 5% CO 2 /95% air) on 150 Â 20-mm tissue culture dishes (Nunc, Roskilde, Denmark) and cultured in Dulbecco's modified Eagle's medium F-12 HAM (Sigma Chemical Co, St Louis, MO, USA) with 10% fetal bovine serum (Sigma) and 50 U ml À1 penicillin and streptomycin. Primary cultured NOKs were used as a normal control as described previously (Kang et al, 2000;Endo et al, 2004).…”

“…The amplified products were analysed by 3% agarose gel electrophoresis to ascertain the size and purity of the products. To confirm the identity of the PCR products, they were cloned into a pCR 2.1vector (Invitrogen) and sequenced as described previously (Endo et al, 2004).…”

“…To screen the sequence variations of the CKMT1 gene, PCR-singlestrand confirmation polymorphism (SSCP) analysis and DNA sequencing analysis were performed as described previously (Uzawa et al, 1995;Endo et al, 2004) (Figure 2A). Nine sets of oligonucleotide primers as summarised in Table 3 were used to amplify the entire coding region (exons 1 -9) of the CKMT1 gene.…”

Evidence for critical role of Tie2/Ang1 interaction in metastatic oral cancer

“…As a negative control, triplicate sections were immunostained without exposure to primary antibodies, which confirmed the staining specificity. To quantify the status of the Tie2 and Ang1 protein expression levels, we used the IHC scoring systems described previously (14,(41)(42)(43)(44)(45). The mean percentages of positively stained cells were determined in at least three random fields at x400 magnification in each section.…”