Amplification and activation of c-Ki-ras oncogene in cell line developed from human pancreas explants.

Amplification and activation of c-Ki-ras gene was studied in normal human pancreas and a cell line (T-3) derived from normal pancreas explants exposed to methylnitrosourea (MNU) for 26 weeks. Normal genomic DNAs from pancreas and derived cell lines showed no transforming activity in NIH 3T3 cells. However, DNAs isolated from tumorigenic cell line derived from MNU treated human pancreas explants transformed NIH 3T3 cells. The hybridization profiles showed that the c-Ki-ras gene was amplified 5 fold in the tumorigenic cells (T-3). The level of mRNA specific to the c-Ki-ras gene was found to be 50-60 fold higher in the malignant cells than in normal human pancreas. These results suggest that higher expression of ras genes is due to gene amplification and/or activation, which is an important step in carcinogenesis.

MNU-induced point mutation and activation of c-Ha-ras oncogene in cell lines derived from human pancreas explants.

Point mutation and activation of c-Ha-ras oncogene was studied at various stages of carcinogenesis in cell lines developed from MNU-treated human pancreas explants. DNAs from normal pancreas and nontumorigenic cell lines showed no transforming activity in NIH 3T3 cells whereas DNA from one of the tumorigenic cell lines transformed NIH 3T3 cells. In this cell line the point mutation was demonstrated to be at codon 12 of c-Ha-ras gene by the loss of an Msp I site. The mutation possibly affected the transcription of c-Ha-ras gene which in turn contributed to the transformation of these cells.

Loss of a Mr 78,000 marker in chemically induced transplantable carcinomas and primary carcinoma of human pancreas.

Toward the identification of steps in the multiphasic process of human pancreas carcinogenesis we have developed a panel of monoclonal antibodies to normal and carcinogen-treated human pancreas cells. One of these, an IgG3 with strong affinity for a membrane-associated Mr 78,000 protein in fetal and adult parenchymal cells, was purified by high performance liquid chromatography, and used for the detection and characterization of tumorigenic stage in human pancreas carcinogenesis. This protein was present on the cell surface of human pancreas explants exposed to methylnitrosourea for up to 4 months and in nontumorigenic cell lines derived from these explants. It was absent in a morphologically transformed subpopulation of cells in explants treated with methylnitrosourea for longer than 4 months, in tumorigenic cell lines derived from these explants, and in primary carcinomas of human pancreas. The presence of this marker in normal pancreas adjacent to tumors, in hyperplastic cells induced by methylnitrosurea and in nontumorigenic cell lines suggests a correlation between the loss of this membrane-associated marker and cell tumorigenicity.

Amplification of c-Ki-ras-2 oncogene sequences in human carcinoma of pancreas.

c-Ki-ras-2 sequences were visualized in paraffin embedded sections from normal adult human pancreases and 24 carcinomas of pancreas by an in situ hybridization technique. A biotinylated 1 kbp EcoRI fragment of pHiHi3 DNA was used as probe and the oncogene was visualized as one or two large grains of reaction products produced in more than 9% of normal pancreas nuclei by streptavidin-peroxidase complex and diaminobenzidine tetrachloride. Its amplification in pancreatic carcinomas was detected as one or more large grains in 54% of the nuclei. In addition, tumor cells showed small nuclear and cytoplasmic grains scarcely seen in normal pancreatic cells. The differential transcriptional activity of this oncogene in cancer cells and the adjacent normal pancreatic cells on the same section was evident in sections from 5 cases where normal pancreas was present.

Modulation of a membrane-associated marker in progenitor cells of human pancreas carcinoma.

For the identification of early steps in the multiphasic process of human pancreas carcinogenesis we have developed a panel of monoclonal antibodies to normal and carcinogen-treated human pancreas cells. One of these, an IgG3 with strong affinity for a membrane-associated 46 kd protein (p-46) in progenitor cells of methylnitrosourea (MNU)-treated human pancreas, was purified by h.p.l.c. and used to study the modulation of this marker in human pancreas carcinogenesis. This protein was undetectable in adult human pancreas, appeared on the cell surface of a small subpopulation of human pancreas explants exposed to MNU, persisted in proliferating cells in hyperplastic foci, and was abundant in anaplastic cells in this model. This marker was associated with cytoplasmic and nuclear membrane of all cells in non-tumorigenic and tumorigenic cell lines derived from these explants. It was present in primary carcinomas of human pancreas but absent in normal pancreas adjacent to tumors. This p-46 marker appears to be organ and species specific since it was undetectable in other normal tissues and their neoplastic counterparts.

Progression in a chemically induced transplantable human pancreas carcinoma.

A methylnitrosourea (MNU)-induced transplantable human pancreas carcinoma was examined, at 3, 12, 18 and 36 months after its development, for growth and invasiveness in nude mice, karyotypic alteration and the evolution of marker chromosomes. Progression in tumorigenicity and invasiveness of cells were evident by a significant increase in tumor diameters produced within 8 weeks by the cells at 36 months as compared to those developed by cells from 3-month-old cell lines. Chromosome analysis at 3 months showed normal 46 XX karyotype in about 80% and minor anomalies in 20% of the cells. At 12, 18 and 36 months, all cells were hyperdiploid with 53-61 chromosomes and several abnormal marker chromosomes. Marker chromosomes showed non-reciprocal translocations, deletions, inversion and isochromosomes. The absence of chromosome 13 from the earlier stage onward may have resulted in the loss of genes which suppress tumorigenicity. The increase in homogeneously staining regions of marker chromosome 3 at later stages appears to parallel the augmentation in tumor growth and mitotic indices.

Visualization of O6-methylguanine in target cell nuclei of dimethylnitrosamine-treated human pancreas by a murine monoclonal antibody.

A murine monoclonal IgG2b with high affinity and specificity for O6-methylguanine was developed. This allowed visualization of this adduct in paraffin-embedded sections by indirect fluorescence or immunoperoxidase methods. The antibody was used to study and compare the formation and persistence of this adduct detected by immunohistochemistry and the presence of 14C methyl by autoradiography in sections from adult human pancreas explants exposed to [14C methyl]dimethylnitrosamine (DMN) and organ-cultured for up to 7 days. O6-methylguanine was detectable randomly and with varied intensity in parenchymal cell nuclei and cytoplasms, 6 h after exposure to DMN. Following RNase treatment, however, only 7.5% of nuclei revealed measurable staining. The positive nuclei decreased to 2.5% within 24 h and remained about the same throughout the 7 days of culture. This correlated well with the change in percentage of positive nuclei for 14C methyl autoradiographic grains which were selectively condensed over the O6-methylguanine-stained nuclei. The data demonstrate a subpopulation of repair-deficient parenchymal cells of human pancreas exposed to DMN.

Evidence for partial exocrine acinar differentiation in experimentally induced pancreatic ductal/ductular cell tumors.

The pancreatic carcinomas induced in Syrian hamsters by a nitroso compound are of a ductal/ductular cell type. Electron microscopic examination of such tumors has revealed the presence of an occasional isolated acinar cell within the tumor. Based on their cellular characteristics, these acinar cells seem to have derived from ductular cell precursors. When we used a monoclonal antibody which specifically recognizes hamster acinar cells, a larger number of cells bearing acinar cell membrane markers were found in many tumors by an indirect immunofluorescence technique. Since most of these positively reacting cells did not exhibit the characteristic cytological features of acinar cells, such as rough endoplasmic reticulum and zymogen granules, differentiation of precursor cells to acinar cells may possibly occur at the cell membrane level. The presence of a few acinar cells within exocrine pancreatic tumors implies that acinar differentiation or retrodifferentiation can occur in pancreatic neoplasms.

Visualization of c-Ki-ras-2 oncogene sequences in human pancreas, a chemically induced transplantable carcinoma, and carcinomas of pancreas by in situ hybridization.

c-Ki-ras-2 sequences were visualized in paraffin-embedded sections from normal fetal and adult human pancreases, a chemically induced transplantable human pancreas carcinoma (PT-1) and three carcinomas of pancreas by in situ hybridization technique. A biotinylated 1-kilobase-pair (kb) EcoRI fragment of pHiHi3 DNA was used as probe and the oncogene was visualized as one or two large grains of reaction products produced by streptavidin-peroxidase complex and diaminobenzidine tetrachloride in more than 9% of normal pancreas nuclei. Its amplification in the chemically induced cell line was detected as one or more large grains in 72% of the nuclei and numerous cytoplasmic grains. The detection of oncogene in normal pancreases and its amplification in PT-1 cells was validated by Southern analysis of EcoRI digests of genomic DNA extracted from normal pancreases and PT-1 cell line. The oncogene was also demonstrated to be equally amplified in two adenocarcinomas and one undifferentiated carcinoma of human pancreas by in situ hybridization.

Hamster pancreas acinar cell post-differentiation antigen detected by murine monoclonal antibody.

For identification of hamster acinar cells, murine monoclonal antibodies to dispersed adult hamster acinar cells were developed. One of these, Ham-Acl, with strong affinity for a membrane-associated adult acinar cell antigen, was purified by HPLC, isotyped as IgG1 and used for the characterization of a species-specific, immunoprecipitable post-differentiation antigen on adult acinar cells. This antigen of 98 kDa was identified on paraffin sections of adult hamster acinar cells by an indirect immunofluorescence technique. It was undetectable in fetal hamster pancreas, but was present on 2-week-old and older hamster acinar cells.

Duct-like morphogenesis of Longnecker pancreatic acinar carcinoma cells maintained in vitro on seminiferous tubular basement membranes.

We have investigated the behavior of dissociated cells of a moderately differentiated Longnecker transplantable pancreatic acinar carcinoma (Longnecker et al., Cancer Lett., 7: 197-202, 1979) maintained in vitro on acellular seminiferous tubular basement membranes of rat testis. The tumor cells, which grow as solid masses in vivo with little organization, undergo organogenesis in vitro into distinct duct-like structures with lumina in the presence of basement membrane scaffoldings. These duct-like structures were formed by flattened epithelial cells, which exhibited poorly differentiated acinar cell characteristics with few or no zymogen (secretory) granules. The cells lining the duct-like structures retained the pancreatic acinar cell specific antigen as determined by indirect immunofluorescence. DNA synthesis did not accompany duct-like organization; however, all of the cells lining these structures continued to incorporate [3H]leucine for up to 4-5 days of culture. They continued to synthesize and secrete amylase, a marker protein of pancreatic acinar cells, into the medium. These results demonstrate that neoplastic epithelial cells of Longnecker pancreatic tumor differentiate into duct-like structures when they come into contact with a basement membrane scaffolding and do not accumulate well-formed secretory granules. This is in marked contrast to the previously reported in vitro differentiation of cells derived from another transplantable rat pancreatic acinar cell carcinoma where the neoplastic cells were fully cytodifferentiated on seminiferous tubular basement membrane without forming duct-like structures but accumulated abundant well-developed zymogen granules (Watanabe et al., Cancer Res., 44: 5361-5366, 1984). Although the basal lamina promotes differentiation of cells of two different pancreatic carcinomas in vitro, we conclude that the in vitro expression of morphogenetic and cytodifferentiation patterns is dependent upon the intrinsic properties of cells of these two transplantable pancreatic tumors.

Identification of erythroid colony progenitors in a subset of human peripheral null lymphocytes devoid of Fc receptors.

This study was aimed at purifying the progenitors of erythroid burst units (BFU-E) from human peripheral blood. Human mononuclear leukocytes from five normal donors were fractionated into several mononuclear cell subpopulations, including null lymphocytes with (null Fc+) and without (null Fc-) receptor for the Fc fragment of immunoglobulin G, through a succession of rosetting procedures and discontinuous Ficoll-Hypaque gradient centrifugations. The fractionated cells were separately cultured for 14 days in plasma clots in the presence of erythropoietin. Among fractionated cell subpopulations large and numerous BFU-E derived colonies grew only from the Fc- null lymphocyte subpopulation. This fraction, representing less than 4% of all mononuclear cells, also contains cells (42 + 11%) capable to differentiation towards the B-cell and plasma-cell lineages. The Fc+ null lymphocytes, representing less than 9% of all mononuclear cells, contained 15.2 + 3.3% cells capable of differentiation toward the T-cell lineage. The whole null lymphocyte subpopulation generated half the number of BFU-E colonies expected from its content in Fc- null lymphocytes. These data demonstrate that the progenitor of erythroid cells (BFU-E) resides in a small heterogeneous null Fc- subpopulation of circulating lymphocytes and suggest that its in vitro differentiation, though generally subjected to inhibitory and enhancing influences from other circulating cell subpopulations, does not necessarily require interaction with other peripheral blood cells.

DNA adduct formation in rat, human and hamster pancreas treated with methylnitrosourea.

The methylation of cellular macromolecules with dimethylnitrosamine (DMN) and methylnitrosourea (MNU) was studied in organ cultured rat, hamster and human pancreatic explants. At concentrations of DMN and MNU that caused similar methylation of protein in human explants DMN caused only 2.6% and 0.3% of the methylation of DNA and RNA that was produced by MNU. The DNA of explants treated with MNU was analyzed. The O6-methylguanine (O6-MeG)/7-methylguanine (7-MeG) ratio was greater in the hamster DNA than in DNA isolated from either rat or human. The time course of removal of methyl adducts from DNA was followed for 6 h after treatment with MNU. No decline in O6-MeG occurred during this period in hamster explants, although there was a decline in the content of 7-MeA and 3-MeA, whereas there was removal of O6-MeG in the DNA from human pancreas explants.

Ductal metaplasia of human exocrine pancreas and its association with carcinoma.

Monoclonal antibodies to cell surface markers of human exocrine pancreas were used to establish the cytotypic expression of cells forming "tubular complexes" in pancreases from six adults without carcinoma and in the nontumorous pancreatic parenchyma of 16 pancreases with carcinoma. These cells manifested duct cell determinants. In general, the presence of cells with duct cell surface markers within the acini corresponded to the normal distribution of centroacinar cells in the 30 control human pancreases (from cadaveric donors); however, foci of abnormal acini were seen in these pancreases independent of or intermingled with the "tubular complexes." The acini in these abnormal areas were formed by a core of cells and cell processes that expressed duct cell determinants. They were partially surrounded by acinar cells and showed slight or no lumenal dilation. While the causative agent(s), the cell(s) of origin, and the regression and/or progression of these lesions are yet to be determined, the replacement of acini by the spectrum of lesions composed of cells with duct cell surface marker is suggested to constitute ductal metaplasia.

Methylnitrosourea-induced carcinoma in organ-cultured fetal human pancreas.

Explants from 12- to 14-week-old human fetal pancreases were organ cultured in a chemically defined medium and cultured for up to 12 months in the presence or absence of methylnitrosourea (MNU). Differentiation of the exocrine pancreas occurred in vitro, and explants cultured in the absence of MNU for 4 weeks or longer revealed normal acinar structures with zymogen granules. Ducts and ductules also developed normally. The undifferentiated tubular structures of the 12- to 14-week fetal pancreas expressed neither ductal nor acinar cell markers. Acinar cell surface marker appeared first after 2 weeks of culture, and the development of centroacinar and ductal cell markers followed 2 to 4 weeks later. MNU-treated explants showed minimal degeneration and necrosis. MNU caused early loss of apical cytoplasm and zymogen granules in acinar cells, resulting in dilation of acinar lumens, concomitant proliferation of cells bearing duct cell markers, and ductal hyperplasia. Enhanced foci of proliferation and carcinoma developed within 3 and 5 months of treatment, respectively. Cells derived from 4- to 5-month MNU-treated explants were tumorigenic in nude mice. Tumor cells revealed a human karyotype and expressed duct cell surface markers.

Alteration of a membrane associated marker in transplantable rat pancreatic acinar cell carcinoma.

A monoclonal murine IgG has been developed which detects a cellular and intracytoplasmic membrane marker expressed only in the rat pancreatic acinar cell. Membrane aberrations in chemically induced transplantable rat pancreas acinar cell adenoma and carcinoma were examined using this IgG. The presence of this membrane marker was assessed by indirect immunofluorescence using paraffin sections of normal pancreas, acinar cell adenoma and transplanted tumors from rats and nude mice. The intracytoplasmic membrane of cells in acinar cell adenoma revealed a much higher intensity of fluorescence compared with normal acinar cells. In contrast there was an almost complete absence of fluorescent staining of the intracytoplasmic membranes of acinar cell carcinoma while their cell membranes showed an intensity of fluorescence far exceeding that of normal acinar cells. These findings suggest a defect in the dynamics of membrane-associated antigen(s) in these cancer cells.

Cells of origin in human gastric neoplasms.

The epithelium of the surface mucosa of the human stomach is demonstrated to share an antigen (HP-DU-1) with human pancreatic ductal cell surface epithelium detectable by a murine monoclonal IgG. This marker was found to be characteristic of the epithelium of gastric surface mucosa and serves to distinguish these cells from the epithelium of gastric glands, the generative cell zone, the parietal and mucous neck cells. The absence of HP-DU-1 was confirmed in the epithelium of the small and large intestines, gall bladder, tracheobronchial trees, urinary bladder, intrahepatic bile ducts, prostatic and salivary glands. This surface marker was used to examine the participation of the surface mucosal cell in hyperplastic, pre-neoplastic and neoplastic lesions of the human gastric mucosa. Gastric hyperplastic polyps and polypoid hypertrophic gastritis were mainly composed of epithelium bearing HP-DU-1 antigens. In contrast epithelial cells of atrophic gastritis, atrophic gastritis with intestinal metaplasia, and adenocarcinoma of the stomach lacked this antigen.

Architecture of human pancreas: implications for early changes in pancreatic disease.

An increased proportion of ductulelike structures, sometimes dilated, occurs in association with pancreatic adenocarcinoma, chronic pancreatitis, cystic fibrosis, and pancreatic ectasia associated with conditions such as uremia. This frequently is interpreted as ductular proliferation, implying origin in ductal elements. Recent animal studies have provided an altered view of pancreatic architecture that is consistent with acinar dedifferentiation leading to the ductulelike structures, or tubular complexes. The architecture of pancreas from organ donors was studied by light and scanning electron microscopy and by wax reconstruction of serial sections. It is concluded that the zymogen granule-containing cells of normal human exocrine pancreas are arranged as branching tubules that vary in diameter and curve acutely. The tubules frequently end blindly to form acinar structures; less frequently they anastomose. This arrangement is consistent with the interpretation that tubular complexes associated with pancreatic disease result from dedifferentiative changes in acinar cells. Tubular complexes may reflect a defect or defects common to pancreatic disease rather than ductular proliferation specific to each one.

Malignant Clara cell line derived from ethylnitrosourea-induced murine lung adenomas.

Lung adenoma induced in Swiss-Webster mice by transplacental exposure to ethylnitrosourea were minced, cultured and carried as cell lines for up to 2 years. The evolution of cell lines was followed every 3 months by ability to grow in nude mice and by morphological and ultrastructural examination of the tumors. Four cell lines, each originating from a single cell inoculum, were cloned. One of these produced mainly papillary tumors in nude mice and ultrastructurally had the morphology of Clara cells.

Monoclonal antibody for identification of human duct cell carcinoma of pancreas.

Monoclonal antibody to duct cell surface marker (HP-DU-1), produced by hybridoma, was shown to be specific to duct cell surface antigen when tested against normal adult and 12-20-week-old fetal human pancreases. There were no common HLA antigens for locus A, B, C and DR among the 10 pancreas donors; nor did HP-DU-1 reveal any affinity for lymphocytes of donors whose pancreases were used as antigen for immunization of BALB/c mice. Seven out of 7 human pancreas adenocarcinomas including a poorly differentiated human pancreatic tumor expressed surface determinants detectable by HP-DU-1. Cell lines CAPAN-1 and CAPAN-2, derived from human pancreatic adenocarcinoma, also revealed the same antigenic determinants.