Kiyohiro Hamatani

Reexpression of RAG-1 and RAG-2 genes in activated mature mouse B cells.

Recombination activating genes (RAG-1 and RAG-2), involved in V(D)J rearrangement of immunoglobulin genes, have been thought to be expressed only in immature stages of B-cell development. However, RAG-1 and RAG-2 transcripts were found to be reexpressed in mature mouse B cells after culture with interleukin-4 in association with several different co-stimuli. Reexpression was also detected in draining lymph nodes from immunized mice. RAG-1 and RAG-2 proteins could be detected by immunofluorescence microscopy in the nuclei of B cells cultured in vitro and in the germinal centers of draining lymph nodes. These findings suggest that RAG gene products play a heretofore unsuspected role in mature B cells.

RET/PTC Rearrangements Preferentially Occurred in Papillary Thyroid Cancer among Atomic Bomb Survivors Exposed to High Radiation Dose

A major early event in papillary thyroid carcinogenesis is constitutive activation of the mitogen-activated protein kinase signaling pathway caused by alterations of a single gene, typically rearrangements of the RET and NTRK1 genes or point mutations in the BRAF and RAS genes. In childhood papillary thyroid cancer, regardless of history of radiation exposure, RET/PTC rearrangements are a major event. Conversely, in adult-onset papillary thyroid cancer among the general population, the most common molecular event is BRAF(V600E) point mutation, not RET/PTC rearrangements. To clarify which gene alteration, chromosome aberration, or point mutation preferentially occurs in radiation-associated adult-onset papillary thyroid cancer, we have performed molecular analyses on RET/PTC rearrangements and BRAF(V600E) mutation in 71 papillary thyroid cancer cases among atomic bomb survivors (including 21 cases not exposed to atomic bomb radiation), in relation to radiation dose as well as time elapsed since atomic bomb radiation exposure. RET/PTC rearrangements showed significantly increased frequency with increased radiation dose (P(trend) = 0.002). In contrast, BRAF(V600E) mutation was less frequent in cases exposed to higher radiation dose (P(trend) < 0.001). Papillary thyroid cancer subjects harboring RET/PTC rearrangements developed this cancer earlier than did cases with BRAF(V600E) mutation (P = 0.03). These findings were confirmed by multivariate logistic regression analysis. These results suggest that RET/PTC rearrangements play an important role in radiation-associated thyroid carcinogenesis.

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, P = 0.022). 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, P = 0.014). 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 (P = 0.039 and P = 0.010, 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.

Human Chromosome 8 (p12→ q22) Complements Radiosensitivity in the Severe Combined Immune Deficiency (SCID) Mouse

The severe combined immune deficiency (SCID) mouse shows two kinds of phenotypic abnormalities, a high radiosensitivity and an abnormal immunoglobulin gene recombination. A genetic study has revealed that a mutation exists in chromosome 16. However, several attempts to isolate the gene responsible for these phenotypes have been unsuccessful. By making use of the characteristics of radiosensitivity, we conducted complementation experiments to identify a human chromosome which contains the responsible gene. Radioresistant cells were selected from the hybrid cells of the SCID mouse and human fibroblasts. Based on this approach, the gene complementing the SCID phenotype was assigned to human chromosome 8 p12-->q22.

CLONING AND CHROMOSOMAL MAPPING OF THE MOUSE DNA-DEPENDENT PROTEIN KINASE GENE

Abstract Severe combined immune deficiency (scid) mice are assumed to have two types of abnormalities: one is high radiosensitivity and the other is abnormal recombination in immunoglobulin and T-cell receptor genes. The human chromosome 8 q1.1 region has an ability to complement the scid aberrations. Moreover, the localization of the subunit DNA-dependent protein kinase [DNA-PKcs] participating in DNA double-strand break repair in the same locus was clarified. In scid mouse cells, the number of DNA-PKcs products and extent of DNA-PK activity remarkably decrease. These observations gave rise to the assumption that DNA-PKcs is the scid factor itself. In order to determine whether the DNA-PKcs gene is the scid gene, we isolated the mouse DNA-PKcs gene and investigated its chromosomal locus by fluorescence in situ hybridization (FISH). Consequently, it became clear that the mouse DNA-PKcs gene existed in the centromeric region of mouse chromosome 16, determined by cross-genetic study, as a scid locus. This finding strongly suggests that mouse DNA-PKcs is the scid gene.

Increased Expression of ras Genes in Non‐Hodgkin's Lymphomas Is not Associated with Oncogenic Activation of Those Genes by Point Mutation

Twenty‐three cases of non‐Hodgkins lymphoma (NHL) were analyzed for expression of ras genes by in situ hybridization utilizing biotinylated DNA probes. Increased expression of Ki‐ras, Ha‐ras and N‐ras genes was observed in 12 cases, 6 cases and 1 case of NHL, respectively. Genomic DNA extracted from these 23 cases of NHL was region‐specifically amplified by means of polymerase chain reaction to examine the presence of point mutations at the 12th, 13th and 61st codons of Ki‐, Ha‐ and N‐ras genes. Dot hybridization assays with appropriate oligonucleotide probes showed no evidence of point mutation in any case of NHL examined. These results indicate that increased expression of ras genes in NHL is not associated with ras gene activation by point mutation.

Thyroid: Papillary carcinoma with inv(10)(p12.1q11.2) ACBD5/RET

Mini review on inv(10)(p12.1q11.2) ACBD5/RET in papillary thyroid cancer (PTC).

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.