Association of neuroendocrine differentiation with neoadjuvant hormone therapy effects in prostatic cancer

Histological therapeutic effects of neoadjuvant hormone therapy (NHT) in prostatic cancer were examined, focusing on the association with neuroendocrine differentiation (NED), using 69 radical prostatectomy cases. The effects of NHT were classified into 3 grades based on the extent of tumor degeneration as observed with hematoxylin and eosin staining. NED cells in the cancer were semi-quantified into 4 grades (negative, 1+, 2+, and 3+) by immunohistochemical staining of chromogranin A (CgA). According to the therapeutic effects, the cases are divided as follows: good response in 26 patients, intermediate in 20, poor in 23. The histological therapeutic effects were significantly weaker in the CgA-positive group than the CgA-negative group (p=0.02). A close relationship between the extent of CgA expression and the histological response was also demonstrated (p=0.007). In the biopsy specimens before NHT, CgA was positive in 46% (32/69) and there was no significant difference in histological therapeutic effects between the positive and negative groups. However, the therapeutic effects were significantly weaker in 22 CgA-positive cases for both biopsy and prostatectomy specimens than in 18 CgA-negative cases for both specimens (p=0.001). In conclusion, although it seems difficult to predict the therapeutic effects of NHT using the biopsy specimens of prostatic cancer, we believe that NED is negatively associated with histological response of prostatic cancer to NHT.     

Malignant transformation of atypical endometrial hyperplasia after progesterone therapy showing germ-cell tumor-like differentiation

A 31-year-old woman was treated for atypical endometrial hyperplasia (AEH) with high-dose medroxyprogesterone acetate (MPA) therapy to preserve fertility. The AEH was found by repeated cytologic and histologic examinations to have completely disappeared with the therapy, but 3 years after her last follow up she required emergency surgery to treat severe genital bleeding. The hysterectomied uterus consisted mostly of poorly differentiated adenocarcinoma, G3 endometrioid type. Minor AEH was present in the exophytic area, in which some glands were cystically dilated. Part of the AEH had transformed into other histologic features with germ-cell-like differentiation, demonstrated by immunohistochemical positive reaction of placental alkaline phosphatase, alpha-fetoprotein, and human chorionic gonadotrophin. Recurrent AEH had undergone malignant transformation, resulting in the development of well- and poorly differentiated adenocarcinoma and tumor exhibiting germ-cell-like differentiation. The patient died of a massive tumor extension 7 months after surgery. The AEH before MPA therapy and the recurrent tumors had genetically different characteristics based on evidence of a loss of heterozygosity, detected at D8S1132 (chromosomal locus, 8q22.1) in the latter but not in the former, by analysis of genetic alterations using microsatellite markers.     

L157X nonsense mutation of the succinate dehydrogenase subunit B gene in a Japanese patient with right paraaortic paraganglioma

Nuclear genes succinate dehydrogenase B subunit and succinate dehydrogenase D subunit, which encode two mitochondrial complex II subunits, are associated with the development of familial paraganglioma (PGL). Succinate dehydrogenase B subunit gene mutation is highly associated with extraadrenal PGL and subsequent distant metastasis. We describe the case of a 29-year-old Japanese man with a 3-year history of hypertension, headache, and palpitation. Endocrinological examinations showed that the patient had elevated levels of catecholamines, and imaging studies revealed a right paraaortic PGL without distant metastases. The PGL was surgically removed. Genetic analysis of the patient showed a heterozygous thymine deletion at position 470 (c.470delT) in exon 5 of the succinate dehydrogenase B subunit gene complementary DNA. This thymine deletion changed TTG (leucine) to TGA (stop codon) at codon 157 (L157X). It remains unclear whether this mutation was associated with PGL malignancy because the patient has had no metastases for the past 3 years. It has been recently reported that L157X is associated with malignant paraaortic PGL. Thus, strict follow-up is required because this succinate dehydrogenase B subunit gene's nonsense mutation (L157X) may be related to the malignancy.     

Generation of a neutralizing human monoclonal antibody Fab fragment to surface antigen 1 of Toxoplasma gondii tachyzoites

A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant surface antigen 1 (SAG1) of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy-chain gene revealed that the closest germ line V segments were VH3-23. The germ line D segment was D1-7, and the closest germ line J segment was JH4. In the light-chain genes, the closest germ line V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09 × 10(-9) M for Tox203 and 2.01 × 10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.     

PTTG is a Secretory Protein in Human Pituitary Adenomas and in Mouse Pituitary Tumor Cell Lines

The pituitary tumor-transforming gene (PTTG) is a homolog of yeast Securin, which arrests the activation of Separin to induce sister chromatid separation in the transition from metaphase to anaphase. Pituitary tumor-transforming gene is also known to induce angiogenesis during pituitary tumorigenesis. It has not been clarified whether PTTG functions as a cytoplasmic or a nuclear protein. Our immunohistochemical study indicated that PTTG is localized in the cytoplasm of pituitary tumor cells. In the present study, confocal laser scanning microscopy (CLSM) analysis of human pituitary adenomas and immunoelectron microscopy of the mouse pituitary cell line, AtT-20, demonstrated the localization of PTTG in the Golgi apparatus and vesicles. Secreted PTTG was detected by immunoblotting from culture medium of mouse pituitary tumor cell lines. Our results suggested that PTTG is a secretory protein produced by pituitary tumor cells. In addition, PTTG may exert autocrine and/or paracrine functions as a newly proposed important pathway for the action of PTTG.     

Prohormone convertases (PC1/3 and PC2) in rat and human pancreas and islet cell tumors: Subcellular immunohistochemical analysis

Prohormons convertase 1/3 (PC1/3; also termed PC1 or PC3) and PC2 are enzymes that activate prohormones by cleaving the pairs of basic amlno acids. This mechanlsm was inltlally Interred lrom the series of several endocrine and neuroendocrine precursor protoh, inciudlng proinsulin and prolusion. To determine the cellular and sub cellular distribution of PC1/3 and PC2 in the rat snd human pancreas, Immunohlstochemistry was performed using polyclonal antlers against mouse PC1/3 (ST-28) and mouse PC2 (ST-29). These studles showed light and dsctron mlcroacoplc co-locailzation of Insulln, PC1/3 and PC2, and the coexistence of glucagons and PC2 In the pancreatic islets. This tendency of colocalizstion was also depicted In one case of human insulin and three cam of human glucagonomas, as well as In rat Insullnomas. in two cases of human Insullnomas, Incomplete processing of proinsulin was suggested by the absence of PC2. At the sub cellular level in the rat pancreatic lslet, the colocalizstion of PC1/3 and insulin, and that of PC2 and glucagons, were observed in the same secretor granules by immunoelectron, microscopy and Image analysis. These studles suggest that PC1/3 and PC2 can functlon with the specifictties In the processing of proinsulin and proglucagon Into their active forms, respectively, in the normal and neoplastic pancreatic islets.     

Case of uterine cervical carcinosarcoma

Carcinosarcoma (CS) is a rare neoplasm that is called a mixed epithelial and mesenchymal malignancy. CS of the uterine cervix is much less common than its counterparts in the uterine corpus. A 61-year-old, gravida 2, para 2 woman, who had undergone menopause 16 years prior to the presentation, was diagnosed with CS of the uterine cervix. A semiradical hysterectomy was carried out on the diagnosis of stage Ib1 cervical cancer. The patient underwent whole pelvic 45 Gy radiation as a postoperative additional treatment, but she died from multiple organ failure by metastasis 17 months after the operation. The tumor protruded from the cervix to the vagina and measured 4.5 x 3.0 cm. Histologically, the tumor was characterized as a squamous cell carcinoma and mesenchymal malignancy, represented by osteosarcomatous components. The stroma was largely composed of atypical spindle-shaped cells, which were immunohistochemically demonstrated to be of epithelial origin. Uterine cervical CS is one of the aggressive malignancies, and squamous cell carcinomas are common epithelial counterparts of cervical CS as well as adenocarcinomas.     

Preparation of a bioartificial kidney using tubular epithelial cells, and an evaluation of Na+ active transport and morphological changes

We intend to develop a bioartificial kidney using tubular epithelial cells and artificial membranes, and to evaluate the reabsorptive function of the confluent layers. Madin-Darby canine kidney (MDCK) cells were cultured on a nucleopore polycarbonate membrane for up to 4 weeks after confluence to examine the influence of culture period on their properties, such as the localization of Na⁺/K⁺-ATPase and active Na⁺ transport. The results were as follows. Ouabain-sensitive Na⁺ active transport declined at 3 to 4 weeks after confluence in each matrix. The localization of Na⁺/K⁺-ATPase indicated depolarization in the cell membrane 3 to 4 weeks after confluence. Prolongation of the culture period increased the formation of an upheaving cell mass after the formation of the confluent monolayer. Scanning electron microscopy revealed fewer microvilli and more flat cells after 3 to 4 weeks of confluency. We conclude that the decline of Na⁺ active transport in the MDCK cells was due to both the formation of multilayers and a decline of cell function throughout the long period of culture following the formation of the confluent monolayers. Further study for selection of membrane material, the extracellular matrix, and species of cells should be continued. Laboratories for Structure and Function Research Department of Physiology     

In vivo and in vitro differentiation of myocytes from human bone marrow-derived multipotent progenitor cells

OBJECTIVE: Recent studies have shown that bone marrow (BM) contains cells capable of differentiating into myocytes in vivo. However, addition of demethylation drugs has been necessary to induce myocyte differentiation from BM cells in vitro, and precise mechanisms of BM cells' conversion to myocytes and the origin of those cells have not been established. We investigated the expression of myogenic markers during differentiation and maturation of myocytes from BM-derived multipotent adult progenitor cells (MAPC) under physiological culture condition. MATERIALS AND METHODS: Frozen BM samples from 21 healthy donors were used as a source of MAPC. To induce myocyte differentiation MAPC was cultured in the presence of 5% FCS, VEGF, bFGF, and IGF-1, and the expressions of myocyte markers were examined at various time points. We also investigated engraftment and differentiation of MAPC-derived myocytes in vivo. RESULTS: Frozen BM-derived MAPC, cultured under the physiological myogenic condition, demonstrated spatial expression patterns of several myocyte markers similar to that of authentic myocyte differentiation. When injected into murine muscles, MAPC treated with the myogenic condition engrafted and differentiated into myocyte marker-positive cells and myotubes in vivo. CONCLUSION: For the first time, we were able to induce myocyte formation from BM cells under the physiological condition in vitro and demonstrated that treating cells with this condition prior to intramuscular injection increased efficiency of engraftment and differentiation in vivo.