Reiko Ito

The presence of BRAF point mutation in adult papillary thyroid carcinomas from atomic bomb survivors correlates with radiation dose

In papillary thyroid carcinogenesis, the constitutively activated mitogen‐activated protein (MAP) kinase signaling pathway caused by a genetic alteration such as RET/PTC rearrangement or mutation of RAS and BRAF genes, is thought to be a major early event. Among these, the recently identified BRAFV600E mutation has been found at high frequency in adult patients with papillary thyroid carcinoma (PTC). However, the association between this mutation and radiation exposure in adult PTC is still unknown. In this study, we examined the BRAFV600E mutation in 64 PTCs among adult atomic bomb survivors in Hiroshima, Japan, comprising 17 nonexposed (0 mGy) and 47 exposed patients who developed the carcinoma after the bombing, and assessed the association of BRAFV600E mutation with clinico‐pathological and epidemiological variables. The median radiation dose in PTCs with the BRAFV600E mutation was significantly lower than that without the mutation (18.5 vs.156.9 mGy, Wilcoxon rank‐sum test, Pu2009=u20090.022). A significant difference was found in the median latency period (years elapsed from atomic bombing to diagnosis) between exposed patients with and without BRAFV600E mutation (29 vs. 21 yr, Wilcoxon rank‐sum test, Pu2009=u20090.014). These findings were further confirmed by logistic regression analysis with BRAFV600E mutation status as a dependent variable and taking into account possible interactions between the variables. We found that the log‐transformed radiation dose and latency period were independently associated with the BRAFV600E mutation (Pu2009=u20090.039 and Pu2009=u20090.010, respectively). These results suggest that involvement of BRAF mutation in thyroid carcinogenesis in exposed people may differ from that in the nonexposed people.

Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens

Recently, in addition to DNA, RNA extracted from archival tissue specimens has become an invaluable source of material for molecular biological analysis. Successful amplification with PCR/RT-PCR is problematic when using amplicons of short size due to degradation of DNA or RNA. We established an improved method for efficient RT-PCR amplification of RNA extracted from archival formalin-fixed, paraffin-embedded tissue by the elimination of RNA modification and the restoration of RNA template activity. Namely, the preheating in citrate buffer (pH 4.0) of RNA extracted from long-term preserved tissue specimens resulted in significantly increased efficiency of RT-PCR.

IMMUNOHISTOCHEMISTRY OF MYOEPITHELIAL CELLS DURING DEVELOPMENT OF THE RAT SALIVARY GLANDS

u2002Using a battery of monoclonal antibodies specific for rat proteins, immunohistochemistry was carried out on the developing myoepithelial cells (MECs) of the rat major salivary glands. The proteins examined were α-smooth muscle actin (αSMA), h1-calponin (calponin), keratin 14 (K14), β subunit of S-100 protein (S-100β), vimentin and glial fibrillary acidic protein (GFAP). The MECs exhibited immunoreactivity for αSMA, calponin and K14, but not that for S-100β, vimentin and GFAP. Immunoreactivity for αSMA appeared in the MECs from the time when the microfilaments were initially deposited in these cells, i.e., at 20 days in utero in the sublingual and submandibular glands and at birth in the parotid gland. Calponin immunoreactivity was seen 1 day earlier than αSMA. The appearance was almost at the same time as the onset of the MEC differentiation in each gland. A small number of the MECs expressed weak K14 immunoreactivity from the time when the acinus-intercalated duct structure was established, i.e., at 21 days in utero in the sublingual gland, at 5 days after birth in the perotid gland and after 5 weeks post-natally in the submandibular gland. In addition, K14 immunoreactivity was observed in the basal cells of the striated and excretory ducts. The first appearance of K14 in these cells again coincided with the emergence of the duct system in each gland, i.e., at 20 days in utero in the sublingual gland, at 21 days in utero in the submandibular gland and at 3 days after birth in the parotid gland. Finally, the MECs in all the glands were found to redistribute as the acini matured. As the acini grew rapidly during the weaning period in the parotid and the sublingual glands, the MECs ceased to surround the acini. Thereafter, they disappeared from the acini in the parotid gland, whereas they reappeared in the sublingual gland. In the submandibular gland, the MECs were confined to the terminal tubules until 4 weeks after birth. Thereafter, the acini were established and invested by the MECs. In conclusion, immunohistochemistry of calponin and αSMA is a useful tool for identification of the MEC during its earliest differentiation, which has hitherto been possible only electron microscopically. In addition, it is suggested that the MEC is heterogeneous and the functionally differentiated MEC appears after weaning around acini of the mucous and seromucous glands.

Expression of Interleukin-6 and Its Effect on the Cell Growth of Gastric Carcinoma Cell Lines

The expression and the effect of IL‐6 were examined in human gastric carcinoma cell lines to determine whether IL‐6 serves as a growth stimulator. The expression of IL‐6 mRNA was detected in three (TMK‐1, MKN‐1, MKN‐7) of 8 gastric carcinoma cell lines. All three cell lines secreted IL‐6 into the culture fluid, in large amounts in the cases of MKN‐1 and MKN‐7 cells. Scatchard plot analysis of IL‐6 binding revealed that MKN‐1 and MKN‐7 cells had both high‐ and low‐affinity receptors. Cell growth of MKN‐1 and MKN‐7 cells was stimulated by IL‐6, while anti‐IL‐6 antibody inhibited growth. The expression of IL‐la mRNA by these three cell lines was induced by IL‐6. IL‐la increased the expression of mRNA for IL‐6 by TMK‐1 cells. These findings indicate that IL‐6 induced by IL‐la is an autocrine growth factor for some gastric carcinomas

Immunohistochemistry of carbonic anhydrase isozymes VI and II during development of the rat salivary glands

Abstractu2002Secreted carbonic anhydrase (isozyme VI; CA VI) was localized by immunohistochemistry in the developing postnatal rat submandibular and parotid glands using a specific monoclonal antibody to the rat enzyme. CA VI immunostaining was not detectable in the glands before birth. In the submandibular gland, granular immunostaining for CA VI was detectable in several terminal tubule cells of 1-day-old rats. At 1 week, the CA VI-positive cells were located at the periphery of the terminal tubules and appeared to be budding off the tubules. These cellular buds gradually increased, and, by 4 weeks, formed acini. CA VI was also detected in the duct lumen from day 1. The immunostaining in the parotid gland was detected sporadically in the acinar cells at 2 or 3 weeks. By 4 weeks, when the gland was almost indistinguishable from the adult one, the number of positive acinar cells had increased. Their number, however, was far smaller than in the adult gland, and the enzyme could not be detected in the duct lumen. CA II was also localized using specific antibodies to the rat isozyme. CA II was detectable in the inter- and intralobular striated ducts at 2 weeks after birth in the submandibular gland and at 3 weeks in the parotid gland. These results suggset that CA VI is secreted into saliva from soon after birth and that CA II appears in parallel with the functional maturation of the ducts. In addition, CA II was transiently expressed by the cellular buds of the submandibular gland at 2 and 3 weeks.

Reduced Expression of Cyclin-Dependent Kinase Inhibitor p27Kip1 in Oral Malignant Tumors

p27Kip1, a cyclin-dependent kinase (CDK) inhibitor, blocks progression from the G1 to S phase by binding cyclin E-CDK2 and inhibiting their activities. We studied the expression of p27 in oral tumors by immunohistochemistry to determine whether lack of p27 plays a role in the development and progression of oral cancer. Reduced expression of p27 was detected in 86% of the squamous cell carcinomas and 95% of the mucoepidermoid carcinomas, respectively, while p27 expression was well preserved in the pleomorphic adenomas. The expression of p27 showed an inverse correlation with the expression of cyclin E in the squamous cell carcinomas and mucoepidermoid carcinomas. However, there was no relationship between clinicopathological parameters and p27 expression. These results suggest that the reduction of p27 protein may confer the development of oral squamous cell carcinoma and mucoepidermoid carcinoma partly through the increased expression of cyclin E.

Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues

Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies.

Abstract B35: Genetic and epigenetic alterations in colorectal cancer among atomic bomb survivors

Colon cancer among atomic bomb survivors has been shown at a significantly high excess risk attributed to atomic radiation exposure. In this study, we focused on microsatellite instability (MSI)-related colorectal carcinogenesis and analyzed both epigenetic and genetic alterations of MLH1 and Ras-signaling related genes, as well as CpG island methylator phenotype (CIMP). Study subjects were 35 colorectal cancer cases from the RERF cohort study (Life Span Study). The MSI-high (H) was found in five cases with use of six microsatellite markers, showing a significantly higher median radiation dose than that of microsatellite stable (MSS) and MSI-low (L) cases. All five MSI-H cases carried LOH of MLH1, three of which had methylated MLH1 in the remaining alleles. Two cases with unmethylated MLH1 were found to have point mutations in MLH1, presumably causing truncated protein and splicing abnormality. Of 30 MSS and MSI-L cases, two methylated MLH1 cases were identified, i.e., a total of five methylated MLH1 cases in all study cases, and these five cases showed a higher radiation dose than that of unmethylated cases. Undetected levels of MLH1 protein expression were confirmed with four of the five MSI-High cases. Next, we analyzed the gene alterations (BRAF and K-RAS mutations and RASSF2 methylation) in the Ras-signaling pathway, which is thought to be a preceding event of MLH1 methylation. All five MSI-H cases carried one or two alterations of these Ras-signaling-related genes, and a total of 18 cases were found to have altered Ras-signaling-related genes with a significantly higher radiation dose than that of 17 cases without the alternations. When the study cases were grouped by the numbers (2, 1, and 0) of alternated Ras-signaling-related genes, three groups revealed a remarkably increasing trend in radiation dose with increased numbers of altered genes. Although CIMP status significantly correlated with MLH1 methylation, no association was found with CIMP status and radiation dose. The results obtained thus far suggest that radiation exposure may influence MSI-related epigenetic/genetic alterations in colorectal carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B35.