Differential Inactivation of Caspase-8 in Lung Cancers

Abstract

© 2 0 0 2 L a n d e s is an apoptosis inducing cysteine protease which is activated through the formation of a death-inducing signaling complex when death receptors are complexed to their specific ligands. Recent reports indicate that CASP8 expression is lost via a combination of promoter methylation and allelic loss in a subset of neuroblastomas. We investigated the state of the gene in lung tumors and cell lines. RT-PCR studies indicated that gene expression was lost in most (27 of 34, 79%) of small cell lung carcinoma (SCLC) cell lines, but expression was retained in all 22 non-SCLC (NSCLC) lines tested. Loss of gene expression at the RNA level was associated with absent protein expression by Western blotting and lack of CASP8 enzymatic activity. Methylation of the promoter region of the CASP8 gene was present in 16 of 27 (59%) of the SCLC lines lacking gene expression. All methylated cell lines lacked the presence of an unmethylated allele indicating biallelic methylation or loss of non-methylated allele. Promoter methylation was absent in all SCLC and NSCLC cell lines retaining gene expression, and all of these lines had the unmethylated form of the gene. One non-expressing SCLC cell line, NCI-H82, had a homozygous deletion at 2q33 encompassing the chromosomal location of the CASP8 gene. The mechanism of gene inactivation in the remaining 10 of 27 (37%) non-expressing SCLC cell lines is unknown. Using five polymorphic markers for 2q33 a high frequency of allelic loss was present in SCLC lines. Analyses of fresh tumors showed that 15 of 43 (35%) of the SCLC, seven of 40 (18%) of bronchial carcinoids and none of 44 NSCLC tumors had CASP8 promoter methylation. Because only approximately 60% of SCLC cell lines lacking CASP8 expression were methylated, extrapolating from the cell line data, we estimate that approximately 58% of SCLC and 30% of bronchial carcinoids lack CASP8 expression. Thus, CASP8 expression is absent in a subset of both high grade (SCLC) and low grade (carcinoid) neuroendocrine lung tumors but not in NSCLC, which usually lack neuroendocrine features. CASP8 may function as a tumor suppressor gene in neuroendocrine lung tumors. B i o s c i e n c e . N o t f o r d i s t r i b u t i o n .

Aberrant methylation of trail decoy receptor genes is frequent in multiple tumor types

“…Amplification of the desired PCR product for each gene was initially confirmed through direct sequencing of the PCR product as described previously. 16 The house keeping gene GAPDH was used as an internal control to confirm the success of the RT reaction. PCR products were analyzed on 2% agarose gels.…”

“…Amplification of the desired PCR product for each gene was initially confirmed through direct sequencing of the PCR product as described previously. 16 Briefly, for MSP analysis 1 g of genomic DNA was denatured by NaOH and modified by bisulfite. The modified DNA was purified using Wizard DNA purification kit (Promega), treated with NaOH to disulfonate, precipitated with ethanol and resuspended in water.…”

Integrins as a distinct subtype of dependence receptors

“…In the case of disseminated neuroendocrine tumors, such as small-cell carcinoma and neuroblastoma, the loss of caspase 8 is associated with malignant disease. 113,114 Importantly, the frequency with which these events are observed in established tumors suggests that the requirement for integrin ligation by the local ECM imposes an important hurdle that must be overcome during oncogenesis (Figure 3).…”

Cisplatin activates Akt in small cell lung cancer cells and attenuates apoptosis by survivin upregulation

“…The intrinsic pathway involves mitochondria that in response to noxious stimuli release the apoptogenic factors cytochrome c and Smac/DIABLO that activate caspases. It has been shown recently [7][8][9] that in SCLC cells components of the death receptor pathway are silenced by aberrant promoter methylation. Due to impaired death receptor signaling the functional integrity of the mitochondrial pathway is crucial for apoptosis induction in SCLC.…”