Hiroshi Kamma

Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer.

Small cell lung cancer (SCLC) is characteristically not associated with tumour‐infiltrating lymphocytes. Since SCLC has been reported to show marked reduction of class I HLA, the reduced expression has been considered a means of escaping anti‐cancer immunity. However, HLA‐DR expressed in cancer cells is now known to contribute to anti‐cancer immunity. To clarify the difference in HLA‐DR expression between SCLC and non‐small cell lung cancer (NSCLC), and the mechanism, the expression and the cis‐ and trans‐acting factors involved were investigated. HLA‐DR was not immunohistochemically detected in any SCLC and could not be induced by interferon gamma (IFN‐γ) in any SCLC cell line, whereas HLA‐DR was expressed to varying degrees and was easily induced in NSCLC. SCLC cell lines lacked class II transactivator (CIITA) even after IFN‐γ induction, whereas NSCLC cell lines expressed CIITA. The other class II HLA‐specific transcription factors were expressed and genomic DNA of HLA‐DR, including the promoter, was conserved well both in SCLC and in NSCLC cell lines. CIITA transfection improved the expression of HLA‐DR in SCLC. In conclusion, the lack of CIITA results in severe deficiency of HLA‐DR expression in SCLC. Since CIITA has also been reported to induce class I HLA, CIITA transfection might make it possible to establish effective anti‐cancer immunotherapy against SCLC through the up‐regulation of class I and class II HLA. Copyright

Complicated Mechanisms of Class II Transactivator Transcription Deficiency in Small Cell Lung Cancer and Neuroblastoma

Small cell lung cancer (SCLC) and neuroblastoma (NB), the most aggressive adult and infant neuroendocrine cancers, respectively, are immunologically characterized by a severe reduction in major histocompatibility complex (MHC) that is indispensable for anti-tumor immunity. We had reported that the severe reduction of MHC in SCLC was caused by a deficient interferon (IFN)-gamma-inducible expression of class II transactivator (CIITA) that is known as a very important transcription factor for IFN-gamma-inducible class II and class I MHC expression (Yazawa T, Kamma H, Fujiwara M, Matsui M, Horiguchi H, Satoh H, Fujimoto M, Yokohama K, Ogata T: Lack of class II transactivator causes severe deficiency of HLA-DR expression in small cell lung cancer. J Pathol 1999, 187:191-199). Here, we demonstrate that the reduction of MHC in NB was also caused by a deficient IFN-gamma-inducible expression of CIITA and that the deficiency in SCLC and NB was caused by similar mechanisms. Human achaete-scute complex homologue (HASH)-1, L-myc, and N-myc, which are specifically overexpressed in SCLC and NB, bound to the E-box in CIITA promoter IV and reduced the transcriptional activity. Anti-sense oligonucleotide experiments revealed that overexpressed L-myc and N-myc lie upstream in the regulatory pathway of HASH-1 expression. The expression of HASH-1 was also up-regulated by IFN-gamma. Our results suggest that SCLC and NB have complicated mechanisms of IFN-gamma-inducible CIITA transcription deficiency through the overexpressed HASH-1, L-myc, and N-myc. These complicated mechanisms may play an important role in the escape from anti-tumor immunity.

hnRNP B1 expression in benign and malignant lung disease

Evidence is accumulating to suggest that hnRNP B1 expression may be a useful tool in the early diagnosis of lung cancer. This study examined the immunohistochemical expression of hnRNP B1 in archived sections of resected lung cancers and compared the patterns of expression with those seen in similar archived sections of non‐neoplastic lung. Particular attention was paid to the expression of hnRNP B1 in the benign bronchial cells in both cases, to establish if overexpression of this protein in respiratory epithelial cells is specific for malignancy. Nineteen cases of different types of non‐small cell carcinoma were examined (eight squamous cell, six adenocarcinomas, two carcinosarcomas, two undifferentiated large cell carcinomas, and one mucoepidermoid carcinoma) and compared with sections from 16 open lung biopsies (three cases of cryptogenic fibrosing alveolitis, two cases of sarcoidosis, two cases of organizing pneumonia, and one case each of tuberculosis, extrinsic allergic alveolitis, non‐specific interstitial pneumonitis, pneumocystis pneumonia, aspergilloma, respiratory bronchiolitis–interstitial lung disease, mineral dust disease, Sjögrens syndrome and systemic sclerosis vascular variant). All the tumours showed positive staining, with the vast majority, 16/19 (84%), showing strong diffuse nuclear staining. The background cells of these cases showed positive staining in alveolar macrophages, lymph node germinal centres, bronchial mucous glands, and bronchial epithelial cells. No significant difference was seen in the percentage of positive bronchial epithelial cells in bronchi adjacent to the tumour compared with the resection margins. In the benign lung cases, positive bronchial epithelial cells were seen in a small percentage, 3/16 (18%), of cases, but the majority of cases showed no or very focal staining. The levels of expression between benign epithelial cells of malignant cases, compared with benign, showed a significant difference when the staining was assessed in percentage of positive nuclei (p = 0.001, Fishers exact test). The results confirm that hnRNP B1 is widely expressed in a range of lung carcinomas; that expression is seen in benign bronchial epithelial cells and inflammatory cells; and that expression in background bronchial epithelial cells appears to be higher in malignant than in benign lung disease. It is feasible that this biomarker may be of use in the detection of early lung cancer, provided that levels of expression can be accurately quantified. Copyright

Expression of MHC class II antigens in human lung cancer cells

SummarySurgical specimens of lung cancers were examined immunopathologically for the expression of major histocompatibility complex class II (MHC-II) antigens in the tumor cells and their relationship to the lymphocytic infiltration. A lymphocytic infiltrate was frequently observed in the tumor tissue, though its intensity differed among the various histological types. MHC-II antigens were often demonstrated in tumors with a lymphocytic infiltrate. They were detected predominantly in the cytoplasm of tumor cells and to a lesser extent on the cell membranes. The emergence of the MHC-II-positive tumor cells was closely related to a local infiltration by lymphocytes including interferon-gamma (IFN-gamma)-producing T-cells. On the basis of the histological findings, an in vitro experiment was carried out. Foutypes of lung cancer cells were incubated with recombinant IFN-gamma in order to induce MHC-II antigens. MHC-II antigens (HLA-DR as well as HLA-DQ and HLA-DP antigens) were elicited in three cancer cell lines depending on the concentration of IFN-gamma. Immunoelectron microscopic study revealed that they were expressed on the surface of the cell membrane, though to a lesser extent than in the cytoplasm. It was considered that MHC-II antigens could be induced in some tumor cells in the immunological environment where IFN-gamma was secreted from T-cells and concentrated locally.

Proliferative activity in thyroid tumors

To clarify the proliferative activity of papillary thyroid carcinoma, the bromodeoxyuridine (BrdU) labeling index (LI) of 61 various thyroid tumors was investigated using an in vitro labeling technique and immunohistochemical staining with anti‐BrdU monoclonal antibody. The mean LI (± standard deviation) of 31 papillary carcinomas, 12 adenomas, 10 adenomatous goiters, 3 follicular carcinomas, and 2 medullary carcinomas were 1.2% (± 1.3%), 0.6% (± 0.3%), 0.7% (± 0.6%), 1.5% (± 2.2%), and 0.7% (± 0.6%), respectively. The LI of the papillary carcinomas ranged from 0.1% to 4.6%, and approximately 66% of these showed less than a 1% similarity with almost all benign tumors. However, the LI of the two malignant lymphomas and the one anaplastic carcinoma were more than 13%. When the LI of the papillary carcinomas was compared with their various prognostic factors, there was no correlation with tumor size, nodal status, or morphologic features. However, the patients who were 50 years of age or older tended to have relatively high LI, and the LI of the papillary carcinomas correlated with patient age. According to these results, the biologic characteristics of papillary carcinomas vary with age and high proliferative activity may contribute to the poor prognosis of this tumor in elderly patients.

Characterization of hnRNP A2 and B1 using monoclonal antibodies: intracellular distribution and metabolism through cell cycle

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 are nuclear RNA binding proteins involved in pre-RNA processing. The alternative splicing of the second mini exon of A2/B1 gene produces A2 and less abundant B1. It has been reported that patients with autoimmune diseases frequently have blood autoantibody valence for A2/B1, and recently that the overexpression, especially of B1, is useful for detecting cancers in early stage. Three anti-A2/B1 monoclonal antibodies were developed using recombinant A2 protein and synthesized peptides around the second splicing site. Three antibodies could separately recognize A2 and B1, and their specificity made them useful in the study of the biochemical and functional properties of A2 and B1. These antibodies have demonstrated differences between A2 and B1 in the intracellular distribution and in the metabolism through cell cycle. They are valuable reagents to clarify the clinical significance of A2/B1 in autoimmune diseases and cancers.

Immunohistochemical study of the hnRNP A2 and B1 in the hippocampal formations of brains with Alzheimer's disease

To elucidate the post-transcriptional regulation in the subjects with Alzheimers disease (AD), we employed immunohistochemical techniques and examined the expression of the heterogeneous nuclear ribonucleoprotein (hnRNP) A2 and B1 in the hippocampus with neurofibrillary tangle (NFT) neuropathology. In the mildly affected subjects (Braak stages I and II), the most intense A2 immunoreactivity was observed in the CA3 to CA1 neurons. In the moderately (Braak stages III and IV) and severely affected subjects (Braak stages V and VI), the CA1 region demonstrated a decrease in the number of A2 immunoreactive neurons and in immunoreactivity in the remaining neurons, while within the CA4 to CA2 in the severely affected subjects, the majority of neurons showed increased A2 immunoreactivity. An intense B1 immunoreactivity was observed throughout the CA subfields. In the CA1 subfield of the moderately affected subjects and in the extensive hippocampal regions of the severely affected subjects, a decrease in B1 immunoreactivity was observed. Double-immunolabeling studies demonstrated that tangle-bearing neurons reduced A2 and B1 immunoreactivity. Our study suggests that hnRNP A2 and B1 display different responses in the AD hippocampus, and further suggests that the post-transcriptional regulation is disturbed in neurons of the AD hippocampus.

Molecular characterization of a mouse heterogeneous nuclear ribonucleoprotein D-like protein JKTBP and its tissue-specific expression

The human DNA- and RNA-binding protein JKTBP is a new member of heterogeneous nuclear ribonucleoproteins (hnRNPs) that are involved in mRNA biogenesis. We cloned and characterized a mouse homolog and studied its expression in mouse tissues. The cDNA encoded a 301-residue polypeptide. There is only a single amino acid difference between the mouse and human sequences. Northern blotting indicated ubiquitous but varied expressions of approximately 1.4 and 2.8kb mRNAs in various tissues. Immunoblotting indicated that the amounts of protein of about 38kDa were higher in the brain and testis than in other tissues. An additional protein of about 53kDa was found in the brain and testis. Germ cell-deficient W/W(v) mutant mice and aged mice had the reduced amounts of JKTBP in the testes. Immunohistochemical staining indicated cell type-specific expression of JKTBP in tissues: neurons and spermatocytes displayed strong signal intensities. The signals were confined to the nucleus. The amount of 38kDa JKTBP was estimated to be approximately 1.3x10(7) molecules per HL-60 cell. These results indicate that JKTBP is an abundant, highly conserved nuclear protein.

Testis- and developmental stage-specific expression of hnRNP A2/B1 splicing isoforms, B0a/b.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and B1 are abundant nuclear proteins that bind to nascent RNAs synthesized by RNA polymerase II. Previously we had found that the splicing isoforms hnRNP B0a/b, from which the ninth exon of the A2/B1 gene is excluded, are abundantly expressed in testis. We postulated that B0a/b are testis-specific isoforms, and investigated the expression of A2/B1 and B0a/b in rat tissues and in postnatal development of rat testes using RNase protection assay, immunoblotting, and immunohistochemistry. We found that hnRNP B0a/b mRNAs are expressed in several tissues but that the testis alone expresses B0a/b proteins. A sequential study using neonatal rat testes demonstrated that B0a/b mRNAs are produced after 17 days of age, but not translated until 4 weeks of age when round spermatids appear in addition to spermatogonia and spermatocytes. Immunohistochemically, hnRNP A2/B1 isoforms are expressed during spermatogenesis from spermatogonia through round spermatids, whereas the expression of A1 is restricted to spermatogonia. This expression pattern in the rat testis is maintained from birth through adulthood. These results suggest that the expression of the hnRNP A2/B1 gene is partly regulated by a testis-specific post-transcriptional mechanism, and that the products of the A2/B1 gene, especially hnRNP B0a/b, are involved in spermatogenesis.

A case of metastasis from gastric cancer to the thyroid gland

Metastatic tumors in the thyroid gland are rarely seen in clinical practice. This report describes a case of metastasis from gastric cancer to the thyroid, found five years after removal of the primary gastric lesion. The patient had a large thyroid mass extending to the mediastinum, but there were no obvious metastatic lesions anywhere except in the thyroid. Subtotal thyroidectomy was performed and histological examination revealed the same findings as those of the original gastric cancer. Of additional interest are the findings that led us to believe this metastatic tumor produced alpha-fetoprotein. Seven months following the operation, the patient died suddenly and, although it is difficult to say whether the patient’s survival was prolonged, we believe that the thyroid surgery gave him considerable palliation.