An SSR-based linkage map of yardlong bean (Vigna unguiculata (L.) Walp. subsp. unguiculata Sesquipedalis Group) and QTL analysis of pod length.

Yardlong bean (Vigna unguiculata (L.) Walp. subsp. unguiculata Sesquipedalis Group) (2n = 2x = 22) is one of the most important vegetable legumes of Asia. The objectives of this study were to develop a genetic linkage map of yardlong bean using SSR makers from related Vigna species and to identify QTLs for pod length. The map was constructed from 226 simple sequence repeat (SSR) markers from cowpea (Vigna unguiculata (L.) Walp. subsp. unguiculata Unguiculata Group), azuki bean (Vigna angularis (Willd.) Ohwi & Ohashi), and mungbean (Vigna radiata (L.) Wilczek) in a BC(1)F(1) ((JP81610 × TVnu457) × JP81610) population derived from the cross between yardlong bean accession JP81610 and wild cowpea (Vigna unguiculata subsp. unguiculata var. spontanea) accession TVnu457. The markers were clustered into 11 linkage groups (LGs) spanning 852.4 cM in total length with a mean distance between adjacent markers of 3.96 cM. All markers on LG11 showed segregation distortion towards the homozygous yardlong bean JP81610 genotype. The markers on LG11 were also distorted in the rice bean (Vigna umbellata (Thunb.) Ohwi & Ohashi) map, suggesting the presence of common segregation distortion factors in Vigna species on this LG. One major and six minor QTLs were identified for pod length variation between yardlong bean and wild cowpea. Using flanking markers, six of the seven QTLs were confirmed in an F(2) population of JP81610 × TVnu457. The molecular linkage map developed and markers linked to pod length QTLs would be potentially useful for yardlong bean and cowpea breeding.

The ACIN1 gene is hypermethylated in early stage lung adenocarcinoma.

INTRODUCTION AND HYPOTHESIS: In recent years, many studies have performed genome-wide searching for differentially methylated genes in cancer. We hypothesized that characteristic aberrant hypermethylation of CpG islands of certain genes may exist in the early stages of lung adenocarcinoma and that such alterations may be useful in the detection and treatment of early lung adenocarcinoma. METHODS: A pair of immortalized cell lines originating from atypical adenomatous hyperplasia (PL16T) and from the resected end of the bronchus of the same patient (PL16B) was searched for aberrantly and differentially hypermethylated DNA fragments by a combination of the methylated CpG island amplification and suppression subtractive hybridization methods. RESULTS: From 229 clones, we selected 15 fragments that had a genomic region meeting the criteria for a CpG island. We identified a gene, apoptotic chromatin condensation inducer 1 (ACIN1), that was hypermethylated in PL16T. A higher frequency of hypermethylation at a locus at the 5': end of the DNA fragment isolated from the ACIN1 gene was found in small-sized adenocarcinoma (2 cm or less) (30/37, 81%) compared with normal lung tissue (9/37, 24%, p < 0.05). Interestingly, hypermethylation of ACIN1 was detected relatively frequently in the normal counterpart of adenocarcinoma without bronchioloalveolar carcinoma (BAC) component (7/16, 44%), but was rare in the normal counterpart of adenocarcinoma with BAC component (2/21, 10%, P < 0.05). CONCLUSIONS: We found hypermethylation of the ACIN1 gene in early stage lung adenocarcinoma. The role of methylation status in the development and malignant transformation of lung adenocarcinoma requires clarification.

Establishment of an immortalized cell line from a precancerous lesion of lung adenocarcinoma, and genes highly expressed in the early stages of lung adenocarcinoma development.

Atypical adenomatous hyperplasia (AAH) is classified as a precancerous lesion of lung adenocarcinoma. We established an immortalized AAH cell line (PL16T) and a human non-neoplastic bronchial epithelial cell line (PL16B) from the same patient by transfection with the gene for SV40 large T antigen. The expression profile of PL16T was compared with that of PL16B by the suppression subtractive hybridization method. From 704 selectively hybridized clones, we finally selected 25 fragments of mRNA that showed transcription levels more than three times higher in PL16T than in PL16B. Thirteen (52%) and eight (32%) of them encoded tumor-associated calcium signal transducer 2 (TACSTD2) and S100 calcium binding protein A2 (S100A2), respectively. The high transcription of TACSTD2 and S100A2 in PL16T was confirmed by in situ hybridization. In normal lung tissue, both TACSTD2 and S100A2 were expressed at very low levels, but seven and five of 14 AAH were positive for TACSTD2 and S100A2, respectively. The frequency of TACSTD2 positivity was increased in 16 of 22 bronchioloalveolar carcinomas (BAC) and adenocarcinoma with mixed subtype with BAC component (mixed BAC). Positivity for S100A2 occurred in four of 22 BAC and mixed BAC. The abnormal transcription of TACSTD2 and S100A2 are thought to be unique molecular markers of the preinvasive stage of lung adenocarcinoma.

Anthracotic index and DNA methylation status of sputum contents can be used for identifying the population at risk of lung carcinoma.

BACKGROUND: Sputum cytology for the mass screening of lung carcinoma is a noninvasive, repeatable, and useful examination, but the detection rate is usually < 0.05% and the reliability is not high. METHODS: The anthracotic index (AI) and methylation status of the promoter regions of the p16, adenomatous polyposis coli (APC), and retinoic acid receptor-beta (RARbeta) genes were examined in 356 sputum specimens after routine cytologic examination. RESULTS: The mean AI of specimens from males was significantly higher than that from females. AI increased with increasing age and smoking index. The mean AI of patients with lung carcinoma was significantly higher than that of the nonaffected population. Furthermore, the mean AI of the specimens with or without cancer cells from patients with cancer was significantly higher than that of the nonaffected population. Abnormal methylation of the p16, APC, and RARbeta genes was detected in 21.7%, 28.2%, and 26.9% of specimens from patients with cancer, respectively. These ratios were significantly higher than those of the nonaffected populations (0%, 3.9%, and 7.6%, respectively). The incidences of abnormal methylation of the three genes were not associated with histologic classification, smoking index, gender, age, or occupation. CONCLUSIONS: These findings suggested that the AI and abnormal methylation status were useful for identifying a population at risk of lung carcinoma using mass screening of cytology specimens.

The implication of background anthracosis in the development and progression of pulmonary adenocarcinoma.

In order to characterize the relationship between background anthracosis and pulmonary adenocarcinogenesis, surgically resected tissues of 66 cases of stage I pulmonary adenocarcinoma, 4 cm or less at their greatest dimension, were examined. These cases were diagnosed based on the classification of small-sized adenocarcinoma of the lung (Noguchi et al., Cancer 75, 1995). Thirteen cases were diagnosed as types A (localized bronchioloalveolar adenocarcinoma, LBAC) and B (LBAC with alveolar collapse), 40 cases as type C (LBAC with a focus of fibroblastic proliferation), 8 as type D (poorly differentiated adenocarcinoma) and 5 as types E (bronchial gland type adenocarcinoma) and F (true papillary adenocarcinoma). The 5-year survival rate of types A and B cases was 100%, while those of type C, type D and types E and F were 52%, 48% and 39%, respectively. Nuclear accumulation of abnormal p53 protein in non-replacement type adenocarcinomas (types D, E and F) was detected more frequently than that in replacement type adenocarcinomas (types A, B and C) (P < 0.05). In each case, black dusty material was extracted from tumorous lesions and non-tumorous regions and blotted onto a nitrocellulose membrane. The anthracotic index (AI) was calculated with a densitometer. AIs of non-tumorous regions in early and replacement type adenocarcinomas (types A and B) were significantly less than in relatively advanced (type C) and poorly differentiated (type D) adenocarcinomas (P < 0.05). These results indicated that adenocarcinoma developing in heavily anthracotic lungs readily progresses to an advanced stage, or that adenocarcinoma with a less favorable prognosis tends to develop in severely anthracotic lungs.