Henry Harris

Hybrid Cells Derived from Mouse and Man : Artificial Heterokaryons of Mammalian Cells from Different Species

Hybrid Cells Derived from Mouse and Man : Artificial Heterokaryons of Mammalian Cells from Different Species

An Abnormal Membrane Glycoprotein Associated with Malignancy in a Wide Range of Different Tumours

Malignancy, as measured by the ability of cells to grow progressively in vivo, is intimately linked to the presence of a structural abnormality in the polysaccharide moiety of one particular membrane glycoprotein. This abnormality is present in a wide range of different tumours; it co-segregates with malignancy in all crosses between malignant and non-malignant cells that have so far been tested; and it remains linked to malignancy in a stringent new test in which non-malignant variants are selected from tumour cell populations by the use of a lectin.

Isoantigen expression in hybrid mouse cells

Abstract Cells of the Ehrlich ascites tumour were fused with A9, A9RI and B82 cells, all three of which were derived from L cells and bore the H-2k histocompatibility antigen complex and the specific surface antigens associated with the presence of the ‘L cell virion’. In the hybrid cell lines derived from these fusions the expression of both the H-2k and the specific L cell surface antigens was suppressed in varying degree. This suppression was demonstrated both by the membrane immunofluorescence tests on the cells themselves and by quantitative absorption tests. Hybrid lines derived from the fusion of Ehrlich ascites cells with diploid fibroblasts explanted directly from CBA-T6T6 mice also showed suppression of the H-2k antigens normally present on the surface of the fibroblasts. When A9 cells were fused with two other ascites tumours, SEWA and MSWBS, which were strain-specific and which bore the H-2s histocompatibility antigen complex, the resultant hybrid cell lines showed no suppression of the surface antigens characteristic of the A9 cell. These antigens were also fully expressed in hybrids derived from the fusion of A9 cells with normal lymphocytes derived from an ASW mouse. The cells of the Ehrlich ascites tumour thus possess the ability to suppress both the histocompatibility antigens and virus-induced surface antigens contributed to the hybrid cell by the other partner. Hybrids between Ehrlich cells and A9 cells, which, on the whole, have a low level of malignancy, occasionally give rise to highly malignant segregants which show marked chromosomal losses. In some of these malignant segregants, but not in others, the H-2k antigen complex contributed by the A9 cell, which was initially suppressed in the hybrid, re-appeared. This demonstrates that the absence of the H-2k antigens in the hybrid cell was due to some suppressive mechanism provided by the Ehrlich component and not to loss of the A9 chromosome bearing the relevant genetic locus. The fact that some highly malignant segregants bear the H-2k antigen complex and some do not indicates that malignancy and histocompatibility segregate independently.

Some Further Information about the Abnormal Membrane Glycoprotein Associated with Malignancy

The abnormal membrane glycoprotein that we previously found to be associated with malignancy in a wide range of different tumours is present in the cell membrane as a dimer and appears to have a specific binding site for glucose.

Ca2 and Ca3 new monoclonal antibodies evaluated as tumor markers in serous effusions

Ca2 and Ca3 are new monoclonal antibodies of IgG1 class, directed against the Ca antigen, a mucustype glycoprotein expressed on the surface of a wide range of malignant human cells and certain specialized normal epithelia. These antibodies were produced by immunization with purified preparations of the Ca antigen. They were tested to assess their value in the diagnosis of malignant effusions. Immuno‐alkaline‐phosphatase staining was used. Smears of pleural and peritoneal effusions were chosen to show: (1) undoubted malignant cells of various types; and (2) mesothelial cells in effusions from cases in which cancer was not in question. The Ca2 antibody, at 1 in 20 dilution of the culture supernatant, was the most specific, giving no reactions with benign mesothelial cells from any of the 35 cases tested. Malignant cells were clearly stained in 35 of 40 cases of carcinoma or mesothelioma. The staining was negative in two cases of oat cell bronchial carcinoma, and in three of four cases of carcinoma of the colon. Ca3 gave similar, but somewhat stronger, reactions with carcinoma cells, but was less specific, reacting weakly with mesothelial cells in 8 of 35 benign effusions. Because the falsenegative reactions given by the Ca series of antibodies are to some extent complementary to those given by monoclonal antibodies directed against the carcinoembryonic antigen (CEA), a combination of Ca2 and anti‐CEA is recommended as a most useful addition to the normal cytologic examination of effusions.

Surface antigens and release of virus in hybrid cells produced by the fusion of a9 fibroblasts with moloney lymphoma cells.

Abstract A9 cells, an 8-azaguanine-resistant subline of the mouse L cell were fused with cells of an immunosensitive Moloney lymphoma (YAC) and with cells of an immunoresistant subline (YACIR). Hybrid cell lines were derived. The hybrid nature of these YAC-A9 and YACIR-A9 cells was confirmed by the presence of chromosomal markers from the A9 cell and H-2.4 (D) surface antigens from the lymphoma cell. Membrane immunofluorescence and mixed hemadsorption tests showed that the hybrid cells express all the surface antigens demonstrable in the parental cells. The difference in the concentration of Moloney-type surface antigen seen in the two lymphoma lines was abolished in the hybrid cells. On the other hand, differences were found in the release of virus by the two hybrid lines, as measured by the ability of the culture medium to infect JLS-V9 indicator cells. Infectious virus was detected in the medium of YAC-A9 cultures, but not in the medium of YACIR-A9 cultures. These findings suggest that the concentration of virus-induced surface antigen and the production of infectious virus vary independently.

Some Thoughts About Genetics, Differentiation, and Malignancy

This article deals with three related questions: (1) whether malignancy is determined by genetic or epigenetic mechanisms; (2) whether epigenetic mechanisms, as conventionally defined, actually exist; (3) what criteria are appropriate for defining dominance or recessiveness of the malignant state in cell fusion experiments.

Complete Suppression of Tumor Formation by High Levels of Basement Membrane Collagen

Suppression of tumorigenicity was first shown in hybrids produced by the fusion of a range of different highly malignant tumor cells with diploid fibroblasts. Cytogenetic analysis of these hybrids revealed that suppression involved a genetic region located in one specific chromosome donated to the hybrid cell by the fibroblast parent. The identity of the gene responsible for this dramatic effect has remained obscure. We now present strong evidence that the primary determinant is the gene specifying collagen XV, a proteoglycan closely associated with the basement membrane. We transfected a line of highly tumorigenic human cervical carcinoma cells with an expression vector carrying the full-length cDNA of the human collagen XV gene. We selected clones making various amounts of collagen XV, examined their growth in vitro, and tested their tumorigenicity in nude mice. High levels of collagen XV altered the growth properties of the cells in three-dimensional cultures. Moreover, we found that, in a dose-dependent manner, the production of collagen XV completely suppressed tumorigenicity in clones that synthesized this molecule at high levels. Immunohistologic studies suggest that suppression is associated with extracellular deposition of the proteoglycan at the cell periphery. (Mol Cancer Res 2007;5(12):1241–5)

Surface antigen expression in malignant sublines derived from hybrid cells of low malignancy

Abstract Ehrlich ascites tumour cells were fused with three L cell derivatives A9, B82 and A9RI, and the surface antigens of the resultant hybrid cells examined. The expression of three surface antigens contributed to the hybrid cells by the L cell derivatives, the H-2 k isoantigen, the virus-induced Moloney-type antigen and the L antigen, was examined by the membrane immunofluorescence and mixed hemadsorption tests. The hybrid cells showed complete or partial suppression of the surface antigens. The hybrid cell lines had a low level of malignancy, but threw off occasional segregant sublines of high malignancy. In these malignant sublines, the three surface antigens reappeared independently of each other. The reappearance of these antigens was accompanied by substancial chromosome losses. This appeared to indicate that the Ehrlich cell possesses mechanisms for the suppression of surface antigens, and that the reappearance of these antigens in segregant tumours results from the loss of the chromosomes determining antigenic suppression.

Collagen XV Inhibits Epithelial to Mesenchymal Transition in Pancreatic Adenocarcinoma Cells

Collagen XV (COLXV) is a secreted non-fibrillar collagen found within basement membrane (BM) zones of the extracellular matrix (ECM). Its ability to alter cellular growth in vitro and to reduce tumor burden and increase survival in vivo support a role as a tumor suppressor. Loss of COLXV during the progression of several aggressive cancers precedes basement membrane invasion and metastasis. The resultant lack of COLXV subjacent to the basement membrane and subsequent loss of its interactions with other proteins in this zone may directly impact tumor progression. Here we show that COLXV significantly reduces invasion of pancreatic adenocarcinoma cells through a collagen I (COLI) matrix. Moreover, we demonstrate that epithelial to mesenchymal transition (EMT) in these cells, which is recapitulated in vitro by cell scattering on a COLI substrate, is inhibited by over-expression of COLXV. We identify critical collagen-binding surface receptors on the tumor cells, including the discoidin domain receptor 1 (DDR1) and E-Cadherin (E-Cad), which interact with COLXV and appear to mediate its function. In the presence of COLXV, the intracellular redistribution of E-Cad from the cell periphery, which is associated with COLI-activated EMT, is inhibited and concurrently, DDR1 signaling is suppressed. Furthermore, continuous exposure of the pancreatic adenocarcinoma cells to high levels of COLXV suppresses endogenous levels of N-Cadherin (N-Cad). These data reveal a novel mechanism whereby COLXV can function as a tumor suppressor in the basement membrane zone.