A radiation-induced murine ovarian granulosa cell tumor line: introduction of v-ras gene potentiates a high metastatic ability.

A non-metastatic epithelial tumor cell line, OV3121, was established from ovarian granulosa cell tumor in B6C3F1 mouse irradiated with 60Co-gamma rays. OV3121 cells showed an epithelial morphology and grew in monolayer with a population doubling time of 28-30 h. The production of estradiol and the expression of cytokeratin confirmed the epithelial origin of the line. No pulmonary metastasis was observed from solid tumors after subcutaneous (s.c.) injection or after intravenous (i.v.) injection of a clonal subline, OV3121-1 cells. We examined the experimental metastasis of individual clones of OV3121-1 cells, containing various introduced viral oncogenes: v-Ha-ras, v-Ki-ras, v-fms, v-mos, v-raf, v-src, v-sis, v-fos and v-myc. Among them, only OV3121-1 cells with v-Ha-MuSV or v-Ki-MuSV produced lung colonies at high frequencies. In a more detailed analysis, the v-Ha-ras transfectants OV-ras4 and OV-ras7 were found to form colonies in various organs by metastasis from tumors after s.c. injection, as well as lung colonies after i.v. injection. Moderately metastatic OV-ras7 cells showed high gelatinolytic activity at 72 kDa (MMP-2) and 92 kDa (MMP-9) as compared with the parental OV3121-1 and OV-Neo control cells by zymographic analysis. However, more metastatic OV-ras4 cells produced progressively weaker bands of 72 kDa gelatinolytic activity. No gross alterations in the expression of MMP-1, MMP-3, TIMP-1 and TIMP-2 transcripts were detected in these cell lines. These results suggest that this ovarian granulosa cell tumor line may provide a useful system for understanding the mechanisms by which oncogenes influence the occurrence of metastasis.

Establishment and characterization of a human gastric scirrhous carcinoma cell line in serum-free chemically defined medium.

We have established a human gastric scirrhous carcinoma cell line (designated as HSC-43) in a serum-free chemically defined medium (CDM) without any polypeptide growth factor, from a primary tumor of a 56-year-old male patient. HSC-43 cells grew in vitro in adherence with a population doubling time of 55 hr, and had the cytological properties of mucinous epithelial tumor cells. Cytogenetic analysis of the cells revealed pseudotetraploidy, with structural abnormalities of deletion at chromosome Iq25 and with 3 marker chromosomes. Some cells had retained features of signet-ring cells and caused fibroblastic proliferation when transplanted into athymic nude mice. The possible involvement of transforming growth factor-alpha (TGF-alpha), and its receptor, the epidermal-growth-factor receptor (EGFR), on the growth of HSC-43 cells was studied. Synthesis and secretion of TGF-alpha by HSC-43 cells were confirmed by biological assay and enzyme-linked immunosorbent assay. Radioreceptor analysis showed the presence of receptors for EGF in HSC-43 cells. Proliferation of HSC-43 cells was inhibited by antibodies against TGF-alpha and/or the EGFR. However, neither TGF-alpha nor epidermal growth factor (EGF) was effective in stimulating the cell growth of HSC-43 cells, irrespective of the cell density when supplemented exogenously. Our data suggest that TGF-alpha and EGFR play a role in the autocrine growth of HSC-43 cells. This may be another example of growth regulation of gastric carcinoma.

Transforming growth factor beta 1 induces apoptotic cell death in cultured human gastric carcinoma cells.

Transforming growth factor beta 1 (TGF-beta 1) is a potent growth inhibitor for many cell types, including tumor cells. We recently have reported the establishment and characterization of two human gastric scirrhous carcinoma cell lines, HSC-39 and HSC-43. Here we examined the effect of TGF-beta 1 on the growth of these lines as compared to five other human gastric adenocarcinoma cell lines. Proliferation of HSC-39 and HSC-43 cells was strongly inhibited by TGF-beta 1, whereas the other gastric adenocarcinoma cell lines were unresponsive to TGF-beta 1. Both HSC-39 and HSC-43 cells gradually lost viability following exposure to TGF-beta 1. This response was dose dependent up to 4 ng/ml. When TGF-beta 1 was removed, the cells failed to exhibit regrowth, indicating an irreversible growth-inhibitory effect of this agent, leading to cell death. DNA fragments were observed consisting of multimers of approximately 180 base pairs 24 h after TGF-beta 1 treatment. The chromatin condensation of each cell line was confirmed by Hoechst 33258 fluorochrome staining. Ultrastructurally, condensed and fragmented nuclei were observed in TGF-beta 1-treated cells. These features are generally associated with apoptotic processes. Both cell death and DNA fragmentation were partially inhibited by cycloheximide, suggesting the requirement for new protein synthesis. Our results suggest that TGF-beta 1 induces cell death in human gastric scirrhous carcinoma cells in vitro which is mediated by activation of a signal transduction pathway for apoptosis.

Establishment and characterization of human signet ring cell gastric carcinoma cell lines with amplification of the c-myc oncogene.

Two unique human signet ring cell gastric carcinoma cell lines (designated HSC-39 and HSC-40A) were established in vitro from the ascites of a 54-year-old male patient. Both cell lines were biologically quite similar, grew in vitro in suspension with a population doubling time of 28-30 h, and had cytological features of mucinous epithelial tumor cells. They formed colonies in soft agar, with a cloning efficiency of 0.8-1.0%. Ultrastructurally, numerous granules were observed in the cytoplasm, suggesting secretory activity. The frequent presence of desmosome and the tight junction at the cell boundary certifies the epithelial origin of the lines. Immunocytochemistry and radioimmunoassay showed production of tumor marker antigens (carcinoembryonic antigen, CA 19-9, and sialyl-Lex-i) and gastrin in both lines. These lines were transplantable in athymic BALB/c nude mice. The histopathology of each line growing in athymic BALB/c nude mice was similar to that of the original tumor. The karyotype of the cells was highly aberrant with structural and numerical changes. The presence of numerous double minute chromosomes and loss of the 13 chromosome and Y-chromosome characterize these lines. In addition, the amplified c-myc oncogene (16-32-fold) was found in both cell lines and original ascitic tumor cells. Overexpression of the c-myc mRNA was noted. These cell lines may be a useful tool, providing both in vivo and in vitro systems for further studies of the biology and therapy of human signet ring cell (or Borrmann's type IV carcinoma) gastric carcinoma.

Reversible squamous cell characteristics induced by vitamin A deficiency in a small cell lung cancer cell line.

A cultured small cell lung cancer cell line (Lu-134-B-S) established from a xenotransplanted tumor in a nude mouse, which had originated from a primary focus of small cell lung cancer, showed morphological changes when the medium was changed from RPMI 1640 supplemented with 10% fetal calf serum to RPMI 1640 supplemented with 10% delipidized fetal calf serum. That is, it consisted of "classic" small cells in the former medium, but after eight passages in the latter medium many cells became squamous cells, possessing abundant eosinophilic cytoplasm and intercellular bridges. Immunohistochemically, they reacted to antikeratin and antiinvolucrin antibodies. Electron microscopically, well developed desmosomes and associated tonofibrils were noted, and electrophoretically, the amount of medium (Mr 57,000 and 59,000) and large-sized (Mr 67,000) keratins were found to increase with the change of the medium. These changes reversed to the original small cell morphology within 4 weeks after addition of vitamin A (retinoic acid) to the medium. These findings suggested that deficiency of vitamin A caused the change of the cell from small to squamous cell and vice versa.

Ciliated-cell adenocarcinoma of the pancreas.

An autopsy case of ciliated-cell adenocarcinoma of the pancreas was reported. A 65-year-old man, who had undergone subtotal gastrectomy because of advanced gastric carcinoma 6 years previously, died of obstructive jaundice. The autopsy revealed a primary tumor in the head of the pancreas and multiple metastatic foci in the liver, lungs, and regional lymph nodes. Histologically, the pancreatic tumor was moderately differentiated papillary adenocarcinoma with well developed cilia. The metastatic tumor also showed similar histology. Histologic and ultrastructural features of the tumor were described in detail and the cytogenesis of ciliated-cell adenocarcinoma was briefly discussed.

Immunohistochemical study of small cell lung carcinoma; with special reference to the neuroendocrine markers aromatic L-amino acid decarboxylase and gastrin-releasing peptide.

Forty-seven surgically resected small cell lung carcinomas (SCLC) were immunohistochemically studied by using antibodies to various neuroendocrine and epithelial markers. SCLC was shown to be subdivided into two categories, with and without the immunoreactive neuroendocrine markers aromatic L-amino acid decarboxylase, gastrin-releasing peptide, serotonin, chromogranin A and neurofilament protein. Neuron-specific enolase (NSE) and creatine kinase BB isoenzyme (CK-BB), which are also considered to be neuroendocrine markers, had a tendency to be widely distributed in the SCLC with a neuroendocrine marker, but the immunoreactivity for both NSE and CK-BB varied in the SCLC without neuroendocrine markers. Therefore they were not included in the classification. Epithelial markers keratin, involucrin and epithelial membrane antigen were frequently observed in the SCLC with neuroendocrine markers, but less so in the SCLC without neuroendocrine markers. The data are discussed briefly in relation to "classic and variant" forms of SCLC in vitro and to a recently proposed histological classification of SCLC.

The frequency of papillomavirus infection in cervical precancerous lesions in Japan: an immunoperoxidase study.

In order to establish the frequency of human papillomavirus (HPV) infection in cervical precancerous lesions of Japanese patients, cervical materials routinely biopsied in the past year were examined immunohistochemically for the papillomavirus genus-specific antigen. Of a total of 832 cervical biopsy specimens, 46 (5.5%) were immunohistochemically positive for HPV. In this study, 206 patients were diagnosed as having dysplasia or carcinoma in situ (CIS), and HPV antigen was found in 15% of these patients. It was found in 13% of patients with mild dysplasia, 28% of those with moderate dysplasia, 17% of those with severe dysplasia and 4.5% of those with CIS. However, HPV antigen was detected only in the epithelium with dysplastic change, not in cancerous areas.

Changes in cell characteristics due to culture conditions in cell lines from human small cell lung cancer.

Eight cultured cell lines were established from human small cell lung cancers. Every cell line showed the morphological and biochemical characteristics of small cell cancer. Changes in cell characteristics were observed in many of these cell lines when culture conditions were changed: "oat cell type" changed to "intermediate cell type" and vice versa when serum-free medium was changed to serum-supplemented medium; a deficiency of vitamin A in the medium caused a change to squamous cells and vice versa; and a tumor promoter (teleocidin B) enhanced the adherence of these cells to the surface of plastic culture dishes. These findings provide evidence that many small cell lung cancer cell lines can change their morphology with changes in the environment of the cells.

High concentrations of enolase, alpha- and gamma-subunits, in the aqueous humor in cases of retinoblastoma.

Because retinoblastomas contain a large amount of the gamma-subunit of enolase or neuronspecific enolase, we studied the aqueous humor levels of the gamma-subunit and alpha-subunit in 12 cases of retinoblastoma. Enzyme immunoassay demonstrated that both the gamma-subunit and the alpha-subunit were greatly increased in all 12 (minimum levels, 87.8 ng/ml and 354 ng/ml, respectively). Thus, aqueous enolase levels, especially that of the gamma-subunit, are useful in the diagnosis of retinoblastoma and may be useful as monitoring markers in retinoblastoma involving the central nervous system.

Morphological variation of immunoreactive cells positive to cholecystokinin 33 (10-20) and gastrin 34 (1-15) in human duodenum.

Human duodenal endocrine cells reactive with antibodies to cholecystokinin (CCK) 33 (10-20) and/or gastrin 34 (1-15) were studied by a combination of immunohistochemical and electron-microscopic methods. By immunohistochemistry, three types of endocrine cells were distinguished in human duodenal mucosa, i.e., those only positive for only CCK, those positive for both CCK and gastrin and those only positive for only gastrin. Ultrastructurally, the first cell type is characterized by many secretory granules with an eccentric dense core (mean diameter; 271 +/- 74 nm). The second cell type, which was less frequent than the other two, has ultrastructural features that resemble type-I cells. The last cell type was composed of two types of cells containing small secretory granules identical to those of IG cells (mean diameter; 171 +/- 31 nm) or large secretory granules indistinguishable from those of I cells (mean diameter; 286 +/- 50 nm).

A case of alpha-fetoprotein-producing rectal carcinoma.

A rare case of rectal carcinoma with a high serum alpha-fetoprotein (AFP) level (maximum, 6,983 ng/ml) is reported. The histology of the primary tumor was adenosquamous carcinoma, the major component being moderately to poorly differentiated adenocarcinoma. Immunohistochemically, a few AFP-positive cells were identified in the primary tumor. However, numerous AFP-positive cells were observed in metastatic tumors in the liver and lungs at autopsy, which were histologically undifferentiated carcinoma with marked pleomorphism. Furthermore, components of adenocarcinoma were present in the metastatic tumors at various sites, and components of squamous cell carcinoma were seen only in the pelvic cavity. To our knowledge, this is the first case of rectal carcinoma with verified AFP production by the tumor cells.

Papillomavirus infection among Japanese: an immunohistochemical study for papillomavirus genus-specific antigen in human surface epithelial lesions.

In order to clarify the relationship between human papillomavirus (HPV) and a variety of surface epithelial lesions, the presence of papillomavirus genus-specific common structural antigen (pgs-antigen) was immunohistochemically investigated in 256 cases of various tumors and tumorous lesions. The pgs-antigen was demonstrated in cases of verruca vulgaris (11/23 cases), condyloma acuminatum (13/26), adult laryngeal papilloma (3/12) and bowenoid papulosis (2/2). No pgs-antigen was observed in ordinary Bowen's disease and other hyperkeratotic skin lesions, such as keratoacanthoma and seborrheic keratosis. In uterine cervical lesions, about 15% of cervical dysplasia, most of which later developed into carcinoma in situ, contained pgs-antigen-positive koilocytotic cells. These results suggest that HPV infection is frequently present in human hyperplastic and atypical surface epithelial lesions of Japanese patients and might indicate possible association with neoplastic transformation, especially in the cervix and skin.

Pancreatic endocrine tumours associated with WDHA syndrome. An immunohistochemical and electron microscopic study.

Nine pancreatic endocrine tumours of patients with watery diarrhoea hypokalaemia achlorhydria (WDHA) syndrome were examined by immunohistochemistry and electron microscopy. All cases revealed neoplastic proliferation of VIP (vasoactive intestinal peptide)-immunoreactive (IR) cells. Immunoreactivity to a novel peptide hormone PHM-27, which is processed from a common big precursor peptide of VIP (prepro VIP/PHM-27), was identified in VIP-IR cells of 8 tumours. VIP-PHM-IR cells had secretory granules measuring about 130 to 220 nm in diameter. Radioimmunoassay of tumour tissue extracts showed high VIP and PHM contents in proportional amounts in most cases. According to the results of immunostaining, the 8 tumours fell into two large groups; 5 with PP (pancreatic polypeptide)-IR cells and 3 with CT (calcitonin)-IR cells. The former group demonstrated VIP cells and PP cells intermingled in various proportions, including one tumour in which coexistence of PP-IR and VIP-IR in the same cells was demonstrated. Cell heterogeneity of the tumours and possible relationships of VIP, PP and CT cells were discussed.

S-100 protein-positive Langerhans cells in various human lung cancers, especially in peripheral adenocarcinomas.

The appearance of S-100 protein-positive Langerhans cells was studied in 90 cases of various lung cancers by an immunohistochemical method. S-100 protein-positive dendritic cells were frequently observed in many adenocarcinomas, especially in those subclassified as bronchiolar cell or type II alveolar cell type. However, no S-100 protein-positive cells were found in "goblet cell type" adenocarcinoma. In some cases of squamous cell carcinoma and large cell carcinoma, these dendritic cells were also observed though they were fewer in number. In all cases of small cell carcinoma, however, S-100 protein-positive dendritic cells were rare. Electron microscopic study of two adenocarcinomas clearly demonstrated many Birbeck granules in the cytoplasm of S-100 protein-positive dendritic cells and confirmed that S-100 protein-positive cells in lung cancer were identical with Langerhans cells.

The levels of three enolase subunits in human tumors; a low ?-/?-subunit ratio as indicator of tumors of neuronal and neuroendocrine nature.

The contents of three subunits (?, ? and ?) of enolase isoenzymes of various human tumors, especially of neuronal and neuroendocrine tumors, were determined by a highly sensitive enzyme immunoassay system. A high level of ?-subunit was detected in neuronal and neuroendocrine tumors, including small cell carcinomas of the lung. The level of ?-subunit was generally higher in all tumors and varied according to the tumor. The ?-subunit level was elevated in rhabdomyosarcomas. Furthermore, analyzing the relationship between the contents of each enolase subunit among the tumors, the ?-/?-subunit ratios were the lowest in neuronal tumors and increased in order of neuroendocrine and non-neuroendocrine tumors. This result suggested that the ?-/?-subunit ratio is an important indicator to evaluate neuronal and neuroendocrine differentiation of the tumor rather than the amount of ?-subunit of enolase itself.

Interconversion of biological characteristics of small cell lung cancer depending on culture conditions.

Two distinct cell lines were obtained from a single heterotransplanted tumor which had originated from a primary focus of small cell carcinoma of the lung (SCCL). They were maintained separately from the beginning in culture media with and without fetal calf serum supplementation. Cells in the serum-free medium grew mostly floating in loose aggregates and showed poor cell cohesiveness, scanty cytoplasm and a few intracytoplasmic small dense-cored granules; all of these features are characteristics of oat cell type SCCL. On the other hand, cells in the serum-supplemented medium grew mostly floating in flatter and more closely associated clumps, were larger, and showed increased cell cohesiveness, occasional tubular structures, better developed organelles including dense-cored granules, and an increased number of cell attachments; these features are characteristics of intermediate cell type SCCL. The modal number of chromosomes differed from each other. Neuron-specific enolase (gamma enolase) and aromatic L-amino acid decarboxylase (ADC) activities in cell pellets were significantly higher in both lines than in control non-small cell lung cancer cell lines. The alpha/gamma type enolase ratio was lower, as was the ADC activity, in serum-free cultures than in serum-supplemented cultures. Interchange of the culture medium induced changes of the growth pattern and cell type from "oat cell type" to "intermediate cell type" and vice versa. The chromosomal number also partially changed. These findings suggest that cultured cells of SCCL alter their growth pattern and cell type depending on the culture conditions and that the selective growth of one cell type might then take place.

Disruptive effect of diethylstilbestrol on microtubules.

Diethylstilbestrol, a unique carcinogen lacking measurable mutagenic potency in Salmonella, was shown to be an inhibitor of microtubule assembly in vitro using microtubule proteins isolated from porcine brains. The effective concentration of diethylstilbestrol was 10-200 microM, as determined by viscometry, turbidity measurement, and electron microscopic analysis.

Enolase distribution in human brain tumors, retinoblastomas and pituitary adenomas.

The cellular localization of alpha-, beta- and gamma-subunits of enolase isoenzyme was studied immunohistochemically in various human brain tumors, retinoblastomas and pituitary adenomas (total, 91 cases). The alpha-subunit was found in almost all brain tumors except in medulloblastoma cases. A case of medullomyoblastoma contained beta-subunit-positive myoblastic cells, which was also positive for human myoglobin. The gamma-subunit was frequently observed in medulloblastomas, retinoblastomas and pituitary adenomas. However, gamma-subunit was also present in some non-neuronal brain tumors such as astrocytomas and oligodendrogliomas. Enzyme immunoassay technique also confirmed the presence of gamma-subunit in a medulloblastoma and two retinoblastomas.