DIFFERENTIATION OF EL4 LYMPHOMA CELLS BY TUMORAL ENVIRONMENT IS ASSOCIATED WITH INAPPROPRIATE EXPRESSION OF THE LARGE CHONDROITIN SULFATE PROTEOGLYCAN PG-M AND THE TUMOR-ASSOCIATED ANTIGEN HTgp-175 Pieter ROTTIERS 1 , Tine V ERFAILLIE 1 , Roland CONTRERAS 1 , Hilde REVETS 2 , Marjory DESMEDT 1 , Hans DOOMS 1 , Walter FIERS 1 and Johan GROOTEN 1 * 1 Department of Molecular Biology, Molecular Immunology Unit, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Ghent, Belgium 2 Department of Immunology, Parasitology and Ultrastructure, Flanders Interuniversity Institute for Biotechnology and Free University of Brussels, Sint-Genesius-Rode, Belgium Progression to malignancy of transformed cells involves complex genetic alterations and aberrant gene expression patterns. W hile aberrant gene expression is often caused by alterations in individual genes, the contribution of the tu- moral environment to the triggering of this gene expression islesswell established. The stable but heterogeneousexpres- sion in cultured EL4/13 cells of a novel tumor-associated antigen, designated as H T gp-175, was chosen for the investi- gation of gene expression during tumor formation. H omoge- neously H T gp-175-negative EL4/13 cells, isolated by cell sorting or obtained by subcloning, acquired H T gp-175 expres- sion as a result of tumor formation. The tumorigenicity of H T gp-175-negative vs. HTgp-175-positive EL4 variants was identical, indicating that induction but not selection ac- counted for the phenotypic switch from HTgp-175-negative to HTgp-175-positive. Although mutagenesis experiments showed that the protein wasnot essential for tumor establish- ment, tumor-derived cells showed increased malignancy, linking HTgp-175 expression with genetic changes accompa- nying tumor progression. T his novel gene expression was not an isolated event, since it was accompanied by ectopic expression of the large chondroitin sulfate proteoglycan PG-M and of normal differentiation antigens. W e conclude that signals derived from the tumoral microenvironment contribute significantly to the aberrant gene expression pattern of malignant cells, apparently by fortuitous activation of differentiation processes and cause expression of novel differentiation antigens as well as of inappropriate tumor- associated and ectopic antigens. Int. J. Cancer 78:503–510, 1998.  1998 Wiley-Liss, Inc. Cancer cells typically feature uncontrolled cell division resulting from mutations in genes that control mitosis, programmed cell death and cell-cell interactions. It is generally accepted that this (cancer) phenotype is acquired gradually, an initial mutation producing a population of precancerous cells. In these cells, the stepwise accumulation of (new) somatic mutations activating cellular proto-oncogenes, such as c-sis, c-erbA and c-myc, or inactivating suppressor genes, such as p53 and RB1, leads to aberrant gene expression and deterioration of intrinsic control mechanisms in growth, differentiation and apoptosis; these pro- cesses are observed in cancer cells and cause their transition to aggressive tumor growth. This complex series of events is referred to as tumor progression. Most somatic mutations found during tumor progression are not indiscriminate. This apparently non- random behavior may reflect the fact that many genetic changes are a disadvantage to the cells and only the relatively few changes that are neutral or that favor the malignant phenotype are retained or selected, respectively. However, increased or decreased gene expression without abnormalities in the encoding DNA may also contribute to tumor progression (Sager, 1997). Analysis of genes expressed differently in tumor cells compared with their normal counterparts revealed more than 500 different transcripts, thus illustrating the extent of the quantitative genetic changes that underlie cancer phenotypes (Zhang et al., 1997). Highly malignant cells may have accumulated relatively few mutated genes but still exhibit changes in expression levels of many genes. These genes encode appropriate but also inappropriate tissue-specific antigens and differentiation antigens, antigens normally expressed during embryogenesis, stress-inducible proteins, as well as macromol- ecules necessary for DNA synthesis, mitosis and cellular structures. Some of these gene products promote tumorigenesis, although they are insufficient to transform a cell. Examples of such progression- enhancing factors are FcRII-B1 (Zusman et al., 1996), the oncofetal carcinoembryonic antigen (Eidelman et al., 1993), cer- tain splice variants of CD44 (Herrlich et al., 1993) and angiogenic factors such as vascular endothelial growth factor (Martiny-Baron and Marme ´, 1995). However, the vast majority of genes that become differentially expressed have no known function in tumor progression; most likely their increased expression is a result rather than a cause of neoplastic transformation. The mechanisms causing overall differential gene expression during tumor progression have been studied extensively. Presum- ably, divergent gene expression arises sequentially, driven by random mutations that directly modulate the expression of the compromised gene. The aberrant expression, whether beneficial or neutral to the cell, will be preserved and will add a new member to the existing set of divergently expressed genes. In addition to this stepwise mechanism, certain positively or negatively acting cancer genes, such as jun, fos, myc and p53, which all encode transcription factors, have been linked to the abnormal expression of whole sets of cellular genes. This simultaneous effect on multiple downstream genes may direct the cell toward a differentiation pathway that is not consonant with the developmental program of the genome. The stepwise accumulation of somatic mutations in individual genes, combined with the downstream activation or inactivation of sets of genes by oncogenes or tumor suppressor genes, appears to account for a major fraction of the differentially expressed genes in tumor cells. Yet the implementation of alterations in these nuclear-acting cancer genes requires translocation of the transcription factor to the nucleus. This translocation may occur in an uncontrolled way or constitutively, due, e.g., to gene translocation (Mitani, 1996), or signaling from growth factor receptors (Perkins et al., 1990) and/or cell adhesion molecules (Behrens et al., 1996). Hence, progression- associated changes in gene expression may also be regulated by normal environmental signals and, exceptionally, may even be reversible. Grant sponsors: Interuniversitaire Attractiepolen and Vlaams Actieco- mite ´ voor Biotechnologie. *Correspondence to: Department of Molecular Biology, K.L. Ledeganck- straat 35, B-9000 Ghent, Belgium. Fax: (+32) 9-264 5348. E-mail: johang@lmb.rug.ac.be Received 3 April 1998; Revised 28 May 1998 Int. J. Cancer: 78, 503–510 (1998)  1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer