Gene expression profiling in undifferentiated thyroid carcinoma induced by high-dose radiation.

Published gene expression studies for radiation-induced thyroid carcinogenesis have used various methodologies. In this study, we identified differential gene expression in a human thyroid epithelial cell line after exposure to high-dose γ-radiation. HTori-3 cells were exposed to 5 or 10 Gy of ionizing radiation using two dose rates (high-dose rate: 4.68 Gy/min, and low-dose rate: 40 mGy/h) and then implanted into the backs of BALB/c nude mice after 4 (10 Gy) or 5 weeks (5 Gy). Decreases in cell viability, increases in giant cell frequency, anchorage-independent growth in vitro, and tumorigenicity in vivo were observed. Particularly, the cells irradiated with 5 Gy at the high-dose rate or 10 Gy at the low-dose rate demonstrated more prominent tumorigenicity. Gene expression profiling was analyzed via microarray. Numerous genes that were significantly altered by a fold-change of >50% following irradiation were identified in each group. Gene expression analysis identified six commonly misregulated genes, including CRYAB, IL-18, ZNF845, CYP24A1, OR4N4 and VN1R4, at all doses. These genes involve apoptosis, the immune response, regulation of transcription, and receptor signaling pathways. Overall, the altered genes in high-dose rate (HDR) 5 Gy and low-dose rate (LDR) 10 Gy were more than those of LDR 5 Gy and HDR 10 Gy. Thus, we investigated genes associated with aggressive tumor development using the two dosage treatments. In this study, the identified gene expression profiles reflect the molecular response following high doses of external radiation exposure and may provide helpful information about radiation-induced thyroid tumors in the high-dose range.

Sam68 is cleaved by caspases under apoptotic cell death induced by ionizing radiation.

The RNA-binding protein Sam68, a mitotic substrate of tyrosine kinases, has been reported to participate in the cell cycle, apoptosis, and signaling. In particular, overexpression of Sam68 protein is known to suppress cell growth and cell cycle progression in NIH3T3 cells. Although Sam68 is involved in many cellular activities, the function of Sam68, especially in response to apoptotic stimulation, is not well understood. In this study, we found that Sam68 protein is cleaved in immune cells undergoing apoptosis induced by γ-radiation. Moreover, we found that Sam68 cleavage was induced by apoptotic stimuli containing γ-radiation in a caspase-dependent manner. In particular, we showed that activated casepase-3, 7, 8 and 9 can directly cleave Sam68 protein through in vitro protease cleavage assay. Finally, we found that the knockdown of Sam68 attenuated γ-radiation-induced cell death and growth suppression. Conclusively, the cleavage of Sam68 is a new indicator for the cell damaging effects of ionizing radiation.

ClpL is a chaperone without auxiliary factors.

Caseinolytic protease L (ClpL) is a member of the heat shock protein (Hsp) 100 family, which is found mostly in Gram-positive bacteria. Here, ClpL, a major HSP in Streptococcus pneumoniae (pneumococcus), was biochemically characterized in vitro. Recombinant ClpL shows nucleotide hydrolase, refolding, holdase and disaggregation activity using either Mg(2+) or Mn(2+) and does not require the DnaK system for chaperone activity. ClpL exhibits two features distinct from other HSP100 family proteins: (a) Mn(2+) enhances hydrolase activity, as well as chaperone activity; and (b) NTPase activity. ClpL forms a hexamer in the presence of ADP, ATP and ATP-γ-S. Mutational analysis using double-mutant proteins mutated at the two Walker A motifs (K127A/T128A and K458A/T459A) revealed that both nucleotide-binding domains are involved in chaperone activity, ATP hydrolase activity and hexamerization. Overall, pneumococcal ClpL is a unique Mn(2+) -dependent Hsp100 family member that has chaperone activity without other co-chaperones.

The ClpP protease of Streptococcus pneumoniae modulates virulence gene expression and protects against fatal pneumococcal challenge.

Streptococcus pneumoniae usually colonizes the nasopharynx of humans asymptomatically but occasionally translocates from this niche to the lungs, the brain, and the blood, causing potentially fatal infections. Spread to other host tissues requires a significant morphological change and the expression of virulence factors, such as capsular polysaccharide, and virulence proteins, such as pneumolysin (Ply), PspA, and CbpA. Modulation of the expression of pneumococcal virulence genes by heat shock and by heat shock proteins ClpL and ClpP, as well as the attenuation of virulence of a clpP mutant in a murine intraperitoneal infection model, was demonstrated previously. In this study, we further investigated the underlying mechanism of virulence attenuation by the clpP mutation. The half-lives of the mRNAs of ply and of the first gene of the serotype 2 capsule synthesis locus [cps2A] in the clpP mutant were more than twofold longer than those of the parent after heat shock, suggesting that the mRNA species were regulated posttranscriptionally by ClpP. In addition, the clpP mutant was defective in colonization of the nasopharynx and survival in the lungs of mice after intranasal challenge. The mutant was also killed faster than the parent in the murine macrophage RAW264.7 cell line, indicating that ClpP is required for colonization and intracellular survival in the host. Furthermore, fractionation studies demonstrated that ClpP was translocated into the cell wall after heat shock, and immunization of mice with ClpP elicited a protective immune response against fatal systemic challenge with S. pneumoniae D39, making ClpP a potential vaccine candidate for pneumococcal disease.

Effect of heat shock and mutations in ClpL and ClpP on virulence gene expression in Streptococcus pneumoniae.

Spread of Streptococcus pneumoniae from the nasopharynx to other host tissues would require the organism to adapt to a variety of environmental conditions. Since heat shock proteins are induced by environmental stresses, we investigated the effect of heat shock on ClpL and ClpP synthesis and the effect of clpL and clpP mutations on the expression of key pneumococcal virulence genes. Pulse labeling with [(35)S]methionine and chase experiments as well as immunoblot analysis demonstrated that ClpL, DnaK, and GroEL were stable. Purified recombinant ClpL refolded urea-denatured rhodanese in a dose-dependent manner, demonstrating ClpL's chaperone activity. Although growth of the clpL mutant was not affected at 30 or 37 degrees C, growth of the clpP mutant was severely affected at these temperatures. However, both clpL and clpP mutants were sensitive to 43 degrees C. Although it was further induced by heat shock, the level of expression of ClpL in the clpP mutant was high at 30 degrees C, suggesting that ClpP represses expression of ClpL. Furthermore, the clpP mutation significantly attenuated the virulence of S. pneumoniae in a murine intraperitoneal infection model, whereas the clpL mutation did not. Interestingly, immunoblot and real-time reverse transcription-PCR analysis demonstrated that pneumolysin and pneumococcal surface antigen A were induced by heat shock in wild-type S. pneumoniae. Other virulence genes were also affected by heat shock and clpL and clpP mutations. Virulence gene expression seems to be modulated not only by heat shock but also by the ClpL and ClpP proteases.