Role of the basic helix-loop-helix transcription factor p48 in the differentiation phenotype of exocrine pancreas cancer cells.

The majority of human pancreatic adenocarcinomas display a ductal phenotype; experimental studies indicate that tumors with this phenotype can arise from both acinar and ductal cells. In normal pancreas acinar cells, the pancreas transcription factor 1 transcriptional complex is required for gene expression. Pancreas transcription factor 1 is a heterooligomer of pancreas-specific (p48) and ubiquitous (p75/E2A and p64/HEB) basic helix-loop-helix proteins. We have examined the role of p48 in the phenotype of azaserine-induced rat DSL6 tumors and cancers of the human exocrine pancreas. Serially transplanted acinar DSL6 tumors express p48 whereas DSL6-derived cell lines, and the tumors induced by them, display a ductal phenotype and lack p48. In human pancreas cancer cell lines and tissues, p48 is present in acinar tumors but not in ductal tumors. Transfection of ductal pancreas cancers with p48 cDNA did not activate the expression of amylase nor a reporter gene under the control of the rat elastase promoter. In some cell lines, p48 was detected in the nucleus whereas in others it was cytoplasmic, as in one human acinar tumor. Together with prior work, our findings indicate that p48 is associated with the acinar phenotype of exocrine pancreas cancers and it is necessary, but not sufficient, for the expression of the acinar phenotype.

Growing colorectal tumors: minimizing microbial and stromal competition and assessing in vitro selection pressures.

Development of primary colorectal cancer cell lines ishampered by contamination from regional microbes, overgrowthof stromal cells, and purported genetic drift from selectionpressures in vitro. We initiated 32 primaryadenocarcinomas, 3 recurrences and 6 distant metastases incell culture. Twelve cell lines from eleven tumors weregenerated (26.8%) overall. Nine of 32 primary tumorsyielded 10 cell lines, 5 were lost to contamination, 13 wereoverwhelmed by stromal cells, and 5 demonstrated no growth.Addition of isobutyl methyl xanthine (IBMX) to culturelimited fibroblastoid growth. There was no associationbetween tumor location (p = 0.535, mid-P), degree ofdifferentiation (p = 0.850, mid-P) or clinicopathologic stage(p = 0.400, mid-P), and the ability of cells to becomeestablished in culture. The majority of cell lines hadsimilar nuclear DNA content and expression of cell-surfaceantigens compared with their parent tumors. Microbialcontamination and stromal cell overgrowth present thegreatest obstacle to capturing a representative bank ofcolon tumors in vitro.

Azaserine-induced rat pancreas tumor model with transplantable cultured cells.

The purpose of this study was to describe the inoculation technique and patterns of growth as well as to characterize typical histological features of Lewis rat subcutaneous and intrapancreatic tumors, induced by inoculation of cultured pancreatic cancer cells (DSL-6A/C1). Subcutaneous inoculation of cultured cells produced a solid tumor that was a locally invasive, well- to moderately differentiated ductal adenocarcinoma. Tumor take was 100% in animals 5 weeks of age; tumor growth was consistent and predictable and a tumor volume of approximately 1 cm3 was reached in 8 weeks. After intrapancreatic transplantation the tumors showed the same histological features as subcutaneous tumors. During inoculation carcinoma cells easily spread around the injected area, and after 2 weeks both pancreatic tumors and superficially infiltrating carcinomas were found in the liver and spleen and around the peritoneum. Tumor take was 60% and tumor growth was somewhat indefinite and unpredictable in the pancreas. However, by reducing the injected carcinoma cell volume and solving the technical problems, 100% tumor take was achieved. The tumor volume reached 2 mm3 during 2 weeks and larger tumors showed a tendency for invasion. According to our results, subcutaneous as well as intrapancreatic tumor induction with cultured cells offers a model for pancreatic cancer studies.

Cell lines derived from pancreatic tumors of Tg(Ela-1-SV40E)Bri18 transgenic mice express somatostatin and T antigen.

Continuous cell lines have been isolated from islet cell, small cell anaplastic and acinar cell carcinomas arising in the pancreas of transgenic mice, line Tg(Ela-1-SV40E)Bri18. These mice carry the pseudogene construct composed of elastase-1 promoter linked to the SV40 T antigen. Cells derived from islet cell or small cell anaplastic tumors secreted insulin and somatostatin during the early period of culture. Phenotypic alterations occurred during culture, whereby insulin secretion ceased and cells instead secreted somatostatin, indicating a change from beta-cell to delta-cell phenotype. Acinar cell lines did not secrete amylase or lipase.

Derivation of ductlike cell lines from a transplantable acinar cell carcinoma of the rat pancreas.

Two cell lines were derived from a transplantable acinar cell carcinoma that had been established from a primary carcinoma of the pancreas in an azaserine-treated Lewis rat. The cultured tumor cells initially produced amylase, but production of exocrine enzymes ceased after 1-2 weeks in culture. The cultured cells were tumorigenic in Lewis rats, and one line produced solid tumors composed of ductlike structures surrounded by dense fibrous tissue. The second cell line produced partially solid and partially cystic tumors with a mixed phenotype of squamous, mucinous, and glandular areas when it grew in vivo following regrafting. Both cell lines lost structural and immunohistochemical acinar cell markers while acquiring duct cell markers during culture and regrafting. These studies provide strong support for the hypothesis that ductlike carcinomas can arise from neoplastic pancreatic acinar cells in rats.

Recent results in animal models of pancreatic carcinoma: histogenesis of tumors.

Animal models of carcinoma of the pancreas provide new information regarding the pathways for histogenesis of the tumors. Four models, induced by chemical carcinogens or transgenic methods, are reviewed briefly from this perspective. Recent reports indicate that carcinomas with a ductal phenotype can arise from transformed acinar cells in rodents. A transgenic mouse model provides evidence that anaplastic carcinomas and islet cell tumors may arise from primitive cells that express the elastase gene, yet retain the potential to differentiate as islet cells. In a nitrosamine-induced hamster model, ductal carcinomas appear to arise directly from ductal cells. Carcinomas in this model contained mutations in the c-K-ras oncogene that are similar to those reported in about 75 percent of human pancreatic carcinomas, whereas acinar cell carcinomas of rats lacked this mutation. The histologic type of a carcinoma may reflect the cell of origin, but this statement is not always true. Therefore, classification of tumors on the basis of phenotype rather than on the presumed cell of origin is recommended. Among the animal models, the carcinomas in hamster pancreas rank as most similar to human pancreatic ductal adenocarcinomas in regard to the phenotype of the tumors and the prevalence of the c-K-ras mutation.

Antiproliferative effect of heparin on human smooth muscle cells cultured from intimal hyperplastic lesions of vein grafts.

The antiproliferative effect of heparin on cultured smooth muscle cells in proliferating human smooth muscle cells derived from clinical lesions of intimal hyperplasia was tested. Smooth muscle cells were obtained from stenotic segments excised from failing in situ saphenous vein bypass grafts in three patients. The nonadventitial portion of the excised tissue was explanted into cell culture using standard techniques without the addition of exogenous growth factors. Under these conditions, rapid cell outgrowth was observed from these explants, in contrast to minimal growth of smooth muscle cells from normal veins from the same patients. Immunohistochemical staining with antiactin antibody confirmed that the cells cultured from the stenotic lesions were smooth muscle cells. Incubation of these cells with porcine mucosal heparin revealed a significant (p less than .01) dose-dependent inhibition of cell proliferation as measured by radioactive thymidine incorporation. Mean inhibition of six subcultures tested ranged from 3 to 46%, at heparin concentrations of 1 to 1,000 micrograms/ml. The magnitude of heparin's antiproliferative effect varied among the cell lines from different patients, but 10-30% inhibition was consistently observed at heparin concentrations usually attained in vivo. The maximal inhibition achieved was 65% in one cell line at the highest heparin dose. We conclude that heparin exerts a significant antiproliferative effect on human smooth muscle cells cultured from intimal hyperplastic lesions from in situ saphenous vein bypass grafts.

Involvement of the RAF1 locus, at band 3p25, in the 3p deletion of small-cell lung cancer.

The ability to establish long-term cell lines of small-cell lung cancer (SCLC) has provided an in vitro model for the disease. We report on the characterization of 10 new human SCLC cell lines established from 34 cytopathologically positive specimens. Based on morphologic and biochemical characterization, growth properties, and expression of MYC and neuroendocrine properties, eight cell lines were categorized as "classic" and two cell lines as "variant". Cytogenetic examination revealed loss of all or part of 3p in all nine SCLC cell lines analyzed. The smallest deletion in common was found at 3p21-3p25. Restriction fragment length polymorphism (RFLP) analyses with probes for 3p were performed for correlation with karyotypic data and supported the cytogenetic findings. In 21 SCLC specimens (cell lines and tumor tissue) with normal DNA, used for comparison, we observed loss of heterozygosity at RAF1 (3p25) in ten of ten informative pairs by using two RFLPs from the RAF1 locus. In addition, loss of heterozygosity was noted in nine of 10 pairs examined with DNF15S2 (3p21) and four of four with D3S3 (3p14). Analysis of cell lines and tumor specimens that lacked paired normal tissue showed a homozygous pattern with the RAF1 probes in all 18 cases. Northern blots revealed significant expression of RAF1 in all cell lines tested. The transcript size was normal. The cytogenetic and RFLP data suggest that the RAF1 locus at 3p25 is involved in the chromosomal deletion of SCLC.

Characterization of preneoplastic and neoplastic lesions in the rat pancreas.

Nodules of acinar cells with increased proliferative potential develop in the pancreas of carcinogen-treated rats and in untreated aged rats. Large nodules are classed as adenomas. Phenotypic and genotypic characteristics of nodule cells were compared with normal pancreas and transplantable acinar cell carcinomas by several methods. Nuclei of acinar cells from normal pancreas, adenomas, and three carcinomas in situ had normal diploid DNA content as determined by flow cytometry. One of two primary carcinomas had a hypodiploid DNA content. Two of three transplantable carcinomas were aneuploid with a DNA content in the tetraploid range. Explants from nodules and adenomas failed to grow in soft agar, whereas several carcinomas were positive in this assay. A primary carcinoma was serially transplanted, but transplantation of nodules or adenomas failed. Transfection of DNA from carcinomas in situ yielded a higher frequency of NIH 3T3 transformants than DNA from adenomas. DNAs from the transformants did not contain ras sequences. These studies indicate that cells from nodules and adenomas have low growth potential and lack critical phenotypic and genotypic characteristics of transformed malignant cells that were present in some primary and transplanted carcinomas.

Abnormalities in structure and expression of the retinoblastoma gene in small cell lung cancer cell lines and xenografts in nude mice.

The putative retinoblastoma gene (Rb) is a tumor suppressor gene which is believed to cause retinoblastomas when both alleles are inactivated, leading to lack of the encoded Mr 110,000-116,000 phosphoprotein. Inactivation of the Rb gene has also been found in several other tumor types, including small cell lung cancer (SCLC). Absence of the 4.7 kilobase mRNA has been found to be frequent in SCLC, and it has been reported that the Rb Mr 110,000-116,000 protein product is always absent, even in tumors expressing Rb mRNA. Using Western blotting technique with a monoclonal antibody directed against the Rb protein, we investigated the expression of the Mr 110,000-116,000 Rb protein in SCLC tumors grown as xenografts in nude mice and/or as cell lines. Rb messenger RNA expression was determined by Northern blotting, and gross structural gene alterations were investigated by Southern blotting. Tumors established from 23 patients were studied. Seven of the tumors did not express Rb protein, whereas expression was detectable in 13. Three tumors were not investigated for protein expression. Only two tumors expressed Rb mRNA without detectable Rb protein expression. Gross DNA alterations were found in four tumors, of which only one expressed Rb mRNA. Our results demonstrated frequent absence of Rb mRNA and protein in SCLC, but apparently normal Rb mRNA and protein were both expressed in more than one-half of the tumors.

Hypodiploid, pseudodiploid, and normal karyotypes prevail in cytogenetic studies of medullary carcinomas of the thyroid and metastatic tissues.

Medullary carcinoma of the thyroid (MCT), often a dominantly inherited neoplasm, derived from intrathyroid C-cells of neural crest origin, is one of the solid tumors least studied cytogenetically. The cells are difficult to grow in culture, only two cell lines having ever been established. Cytogenetic studies of only 5 tumors have been reported previously. In this paper we report on the cytogenetic analyses of 8 specimens of primary and/or metastatic MCT tumor tissue from 6 patients with familial disease, including more recent metastatic tumors in lymph node and femur of a patient whose thyroid and earlier lymph node metastases were described previously. Some of these specimens were harvested sequentially over time. Hypodiploid or diploid modal numbers prevailed with normal, pseudodiploid, or hypodiploid karyotypes.

Molecular mapping of deletion sites in the short arm of chromosome 3 in human lung cancer.

We used 10 restriction fragment length polymorphism (RFLP) probes spanning the length of the short arm of chromosome 3 (3p) to map deletion sites in human lung cancer. Two approaches were used. 1) When a patient's tumor and normal tissue were available, loci with allelic heterozygosity in the normal tissue were tested for loss of alleles at 3p. 2) When the corresponding normal tissue was not available, the frequency of heterozygosity at each locus in a panel of tumors was compared to the corresponding published frequencies in nontumor tissue of healthy individuals or patients with lung cancer. In 14 small cell lung carcinomas (SCLC) with normal DNA for comparison, allele loss was found at all heterozygous loci, with one exception at a locus near the 3p centromere (D3S4). In the total of 53 SCLCs, which included tumors without paired normal tissue, frequency of heterozygosity was significantly reduced in all 10 3p loci. Three loci, DNF 15S2, RAF1, and D3S18, were homozygous in all tumors in the SCLC panel. These loci, which are in regions 3p21 and 3p25, may thus be involved in the origin or evolution of SCLC. We also investigated 24 non-SCLC tumors. In this panel, frequency of heterozygosity was significantly reduced at seven of the 10 loci tested. Comparison of the results shows that the pattern of allele loss on 3p is different in SCLC and non-SCLC, suggesting a difference in pathogenesis at the genetic level.

Mapping and characterization of an X-linked processed gene related to MYCL1.

A DNA sequence with homology to the myc family of proto-oncogenes has been characterized and found to be a processed gene related to L-MYC (MYCL1). This processed gene (MYCL2) was isolated by cross-hybridization to an oligonucleotide probe synthesized from the C-MYC (MYC) sequence in a highly conserved region of the myc gene family. Sequence analysis of MYCL2 revealed an open reading frame of 1194 bp with no intervening sequences and strong homology to the recently published DNA sequence of MYCL1. Southern and Northern blot analyses of DNAs and RNAs from small cell lung carcinomas confirmed its MYCL1 homology. Mapping of MYCL2 by somatic cell hybrids places this sequence on the long arm of the X chromosome in bands q22----q28.

Loss of heterozygosity in a gene coding for a thyroid hormone receptor in lung cancers.

The ERBA beta gene codes for a DNA-binding thyroid hormone receptor (THR) and maps to chromosome 3p21-p25, overlapping a 3p deletion characterizing small-cell lung carcinoma (SCLC). A DNA clone detecting an RFLP at the ERBA beta locus has been used to probe a large number of lung tumors. Virtually all SCLC had lost heterozygosity, showing that the 3p deletion in SCLC includes this gene. A substantial but smaller proportion of non-small-cell carcinomas had lost heterozygosity at ERBA beta. Among all non-small-cell tumors some had lost heterozygosity at the proximal locus DNF15S2 (band 3p21) but not at ERBA beta, whereas none were found where the reverse was true. Therefore, the locus which plays a role in non-small-cell tumorigenesis probably lies closer to DNF15S2 than to ERBA beta and is almost certainly not the latter.

Cell culture of human metastatic breast carcinomas: a review.

The literature concerned with culture of primary and metastatic breast carcinomas is reviewed historically and summarized in the tables. The development of cell culture methodology for these tissues and its usefulness is discussed in conjunction with the problems encountered in culture of breast carcinomas.

Molecular analysis of the short arm of chromosome 3 in small-cell and non-small-cell carcinoma of the lung.

Previous studies have suggested that the loss of DNA sequences on the short arm of chromosome 3 (3p) is associated with small-cell lung carcinoma. We therefore looked for loss of 3p alleles in tumor tissue from 42 patients with either small-cell or non-small-cell lung carcinoma. All 13 patients with small-cell lung carcinoma who were heterozygous for one or more alleles at 3p in normal tissue had the loss of at least one codominant allele in the tumor tissue. Cell lines of small-cell lung carcinoma from an additional eight patients were homozygous for 3p alleles; this result was significantly different from the predicted frequency of homozygosity. The tumor tissue studied included cell lines of small-cell lung carcinoma obtained from biopsy specimens, an autopsy sample, and an excised lymph node containing tumor cells. Loss of alleles at 3p was observed in tumor samples obtained before and after chemotherapy. Four of 15 evaluable patients with non-small-cell carcinoma of the lung had loss of 3p alleles. We conclude that loss of alleles at 3p is a change found consistently in small-cell lung carcinoma and occasionally in non-small-cell lung carcinoma.

Expression of myeloid and major histocompatibility antigens on small cell carcinoma of the lung cell lines analyzed by cytofluorography: modulation by gamma-interferon.

Recent reports have described the expression of myeloid cell surface antigens on cells of small cell carcinoma of the lung (SCCL). In order to confirm and extend these findings, we have examined by cytofluorography a large panel of well-characterized cell lines derived from SCCL tumors for the expression of several myeloid cell-associated antigens, some of which were not examined in previous reports. In addition, we have studied the expression of Classes I and II HLA antigens on these SCCL cell lines. Finally, we examined the effect of gamma-interferon on the expression of several cell surface markers and on proliferation of SCCL cells. We have found that several SCCL cell lines expressed a Mr 55,000 polypeptide antigen, My23, previously found only on monocytes and monocytic leukemia cells. In addition, the cell lines studied expressed another antigen, defined by monoclonal antibody AML-1-99, which is associated with monocytes and hematopoietic stem cells. We confirmed previous studies that the Leu-7 antigenic determinant is expressed on SCCL cells but observed only minimal or absent binding of monoclonal antibody OKM1 to most cell lines. Class I HLA antigens were present on eight of nine lines examined while Class II HLA was expressed on three of nine lines. Gamma-Interferon decreased the proliferative rate of all lines examined. However, this lymphokine was capable of inducing Class I HLA on several lines. The effect of gamma-interferon on other cell surface antigens was variable. These studies confirm that some myeloid cell-associated antigens are expressed on cultured SCCL lines and, additionally, show that their expression can be modulated by immune interferon. Determining the significance of finding myeloid cell-associated antigens on SCCL cells will require further study.

Biosynthesis of procalcitonin in small cell carcinoma of the lung.

Immunoreactive calcitonin (CT) secreted by DMS 53, a cell line derived from human small cell carcinoma of the lung, consists almost entirely of molecular species larger than the mature hormone (Mr 3,420). Messenger RNA isolated from DMS 53 cells and nude mouse tumors was translated in wheat germ systems, and the products were precipitated with CT-specific antisera. Analyses of the translation products by electrophoresis on 15% polyacrylamide-sodium dodecyl sulfate gels indicated synthesis of a Mr 16,500 preprohormone that was reduced to Mr 14,500 by cotranslation with microsomal membranes. Immunoprecipitation of CT from media from pulse-labeled cultures revealed two major products (Mr 16,500 and Mr 14,500) and up to three minor secreted polypeptides (Mr 9,400, 8,400, and 6,800). Intracellular CT from cell homogenates appeared almost entirely as a single major product (Mr 14,500) and possibly 3-4 minor components (Mr 16,500; 9,200, 8,400, and 6,800). No glycosylated forms of CT were demonstrable by lectin binding methods or labeling attempts with tritiated sugars. The presence of multiple CT species in DMS 53 cells suggests significant post-translational processing of the larger precursor molecules and the accumulation and secretion by small cell carcinoma of the lung of several intermediate immunoreactive forms via a glycosylation-independent secretory pathway.

Cytogenetics of medullary carcinoma of the thyroid.

Medullary carcinoma of the thyroid (MCT) is a dominantly inheritable neoplasm derived from intrathyroid C cells. The cytogenetics of this tumor has been only sparsely and indirectly studied previously. This article describes the chromosomes of primary MCT tumor tissue cultured with colcemid for 48 hr, metastatic tumor in lymph node cultured for 7 days from the same patient, and of primary tumor tissue cultured for 3 weeks from a second patient. The modal numbers were 42-44 in all 3 specimens. Karyotypes from the metastastic tumor were similar to those of the primary tumor. Karyotypes of the two primary tumors, however, differed from each other, having in common only the modal numbers and the loss or structural alteration of a #22.

Immunocytochemistry: its evolution and criteria for its application in the study of epon-embedded cells and tissue.

The word immunocytochemistry is currently used to describe a number of methods that can be employed to localize antigens within cells by means of antigen-specific antibodies. In this article we will review a number of these methods, including immunofluorescence, immunoperoxidase, avidin-biotin, and colloidal-gold techniques. The advantages and disadvantages of the various methods are discussed, special attention being focused upon immunocytochemical staining of plastic-embedded tissue. Studies on the light microscope level show that embedding tissue in plastic prior to immunoperoxidase staining not only improves visualization of antigen-specific staining but also provides an accurate and efficient means of prescreening tissue for antigen prior to immunocytochemical staining on the electron microscope level. Varying section thickness between 1 and 3 microns does not significantly influence staining, whereas the fixative used to preserve the tissue under study does. On the electron microscope level, the colloidal gold technique appears superior to immunoperoxidase staining. It is both esthetically more pleasing and highly sensitive. Of five different colloidal gold methods tested, the most sensitive is the two-step technique that employs an antigen-specific primary antibody followed by a gold-labeled secondary antibody. Throughout this article, special emphasis is placed on the use of proper controls, both on the light and electron microscope levels. Where possible, such controls should include substitution of specific antiserum with normal serum; the use of antigen-adsorbed antiserum; the use of antisera with specificities for antigens not present in the tissue being studied; the use of tissue previously shown to be stainable for the antigen; and if cultured cells are being studied, the use of a number of cell types that do not contain the antigen.