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Ad.1

In a recent paper Klein G. Cancer, apoptosis, and nonimmune surveillance, Cell Death and Differentiation, in press, I have briefly summarized this field as follows:

All organisms have powerful surveillance mechanisms that prevent the outgrowth of potentially cancerous cells. Many of them are highly conserved by evolution.

Believed for a long time as the most important safeguard,  immunological surveillance   now occupies a relatively minor place. It is still a powerful restraint against the outgrowth of virally transformed cells, however. This is the reason why tumorigenesis by Epstein-Barr virus, the papillomaviruses, and HHV-8, the Kaposi sarcoma herpesvirus, is either restricted to or more pronounced in the immunodefectives.

Non-immune surveillance is of four different kinds:

i).  Genetic surveillance   is largely based on DNA repair. It is the first line of defense, robustly built on a multitude of repair mechanisms. Defects in repair enzymes lead to specific cancer syndromes, several om them associated with multicancer families.

ii). The evidence for  epigenetic surveillance   is not yet firmly established. Preliminary evidence indicates the existence of inherited differences in the stringency of imprinting, possibly related to cancer risk.

iii).  Intracellular surveillance   prevents the outgrowth of cells driven by illegitimately activated oncogenes. Growth arrest and apoptosis are its two main arms. They are related but distinct. Growth arrest can be assigned to specific tumor suppressor genes, some of which are also linked to apoptotic pathways. Apoptosis is a firmly built multipathway system, as tightly controlled as cell division. Tumor development includes impairment or damage to one or several apoptotic pathways. Tumor cells use multiple escapes from apoptosis, including both the debilitation of proapoptotic pathways (e.g. p53) and activation of antiapoptotic mechanisms e.g. AKT/PI3K. Nevertheless, no tumor cell is completely resistant to apoptosis. Genotoxic agents such as irradiation or chemotherapy, act by inducing apoptosis in relatively apoptosis resistant cells.

Many therapeutic approaches, including clinical trials, aim at the reactivation of apoptosis. Partial of full restoration of a pathway that has been damaged in a given tumor cells is most likely to succeed. The Achilles heel approach involves mRNA or protein targeting, to reduce abnormally high enzyme levels and/or amplified oncoproteins.

iv).  Intercellular surveillance   is less well explored. It is clear, however, that loss of contact by epithelial cells leads to anoikis, a special form of apoptosis. Inhibition of tumor growth by adjacent normal cells is another phenomenon of great interest, largely unexplored by molecular methods. It may explain long range dormancy and counteract the outgrowth of disseminated tumor cells.

Ad.2

Our analytical work in this area focuses on antiapoptotic sculpting. Burkitt lymphoma (BL) has been one of our favorite tools for study during more than three decades. We were among the first to discover the viral (EBV) association of the high endemic African form, the crucial role of the Ig/myc translocation, the nature of the immune response against the tumor and some components of its relative resistance to apoptosis.

As a tumor driven by the myc oncogene the BL cell must acquire a relative resistance to apoptosis during its initial development. Several genetic and epigenetic changes were detected that contribute to this already in the in vivo tumor and in derived lines. The p53 pathway is crippled by either p53 mutation, ARF deletion or MDM2 amplification. The Rb pathway is usually impaired by p16 methylation. Nevertheless, the classical histology of the tumor  referred to as the 'starry sky' appearance  attests to ongoing apoptosis. The white 'stars' are macrophages containing remnants of apoptotic nuclei, derived from the lymphoma cells. Moreover, the tumor is exquisitely sensitive to chemotherapy that acts largely, if not entirely, through the induction of apoptosis.

These and many other findings show that apoptosis resistance is relative rather than absolute. Complete resistance is probably never achieved.

Our own work on BL relates to this problem in several ways. It intersects with a question we have pursued since we and others first showed that 98% of the high endemic (African) BLs carried the virus. In contrast to transformed normal B cells (immunoblasts) that carry the virus in vitro and also the immuneblastomas that arise in immunodefective persons, where the virus expresses its full growth transformation program that includes proliferation driving and antiapoptotic virus-cell interactions, BL cells do not express the growth transformation program. They only express one viral protein, EBNA1, required for the maintenance of the viral genomes in the free episomal form, and two small double stranded RNA molecules, the EBERs.

Cell growth is driven by the constitutively activated myc gene, apoptotic pathways are impaired, as already mentioned, in contrast to EBV transformed immunoblasts where they are not found. But what is, then, the role of EBV in BL?

As mentioned, Csaba Kiss and Laszlo Szekely have discovered that loss of EBV from three different BL lines is accompanied by the downregulation of the tcl-1 oncogene that can be reactivated again by EBV infection. tcl-1 is a powerful activator of the AKT pathway which is, in turn, a major antiapoptotic force. It is therefore likely that EBV contributes to the genesis of BL by conveying additional mechanisms of apoptosis resistance on the myc-driven BL cell.

it may also be noted that the Ig/myc translocation that occurs as an accident during physiological Ig-rearrangement pushes the cells towards an apoptotic or proliferative pathway at a point where they would be normally programmed to go to the resting state and turn into a long lived memory cell, or a plasma cell. It is therefore understandable that the main Ig/myc translocation carrying tumors, human BL, as well as mouse and rat plasmacytoma, depart from exactly these stages in the B cell life cycle. For the human BL cell, it has to be added that the long lived memory B cell is the site of latently persisting EBV. We have shown that the latently infected cells express the same minimalistic EBV program as the BL cells. The postulated apoptosis protection by latent EBV may thus act at a crucial point of apoptotic rescue, and thereby tip the balance towards proliferation.

Ad.3

Ever since the early 50s, my and my collaborators research work has revolved around multistep carcinogenesis. In the early 50s, we have been among the first to document the role of Darwinian variation and selection in the evolution of tumor cell populations. The ability of originally solid tumor cells to grow in the free suspension form of ascites tumors and the development of resistance against cytostatic drugs were the first phenotypic markers we studied in this context. Later we showed with Henry Harris in Oxford, that normal cells could suppress the tumorigenicity of even highly malignant partners in somatic hybrids derived by cell fusion. The suppressor genes, or, as we prefer to call them nowadays, the tumor antagonizing genes that were responsible for these effects could be localized to specific chromosomes.

An unusual feature of these somatic hybrid experiments was the identification of genetic components that suppressed in vivo tumorigenicity, but not in vitro growth

The work of several groups, including our own, pointed to the short arm of human

chromosome 3 (3p) as an important site of tumor antagonizing genes. Two different groups

pursue this area: Stefan Imreh and the group of Eugene Zabarovsky with which we still

collaborate. The more cytogenetic and more molecular approaches of the two groups are

mutually complementary. Recently, Stefan Imreh, Eugene Zabarovsky and I wrote a joint

review of this work ( Imreh, S., Klein, G., Zabarovsky, E.R. Search for unknown tumor

antagonizing genes. Genes, Chromosomes and Cancer, in press.).

The following conclusions are conceptually important:

We have set out to find one or a few crucial tumor suppressor loci on human 3p. Instead, we have identified several genes with proven or suspected tumor antagonizing activity. Over the relatively short 13.5 Mb strech of 3p21.3 more than 60 genes have been identified in four subregions. 29 among them are interesting tumor inhibitory candidates. Taken together with the large number of deletions and heterozygosis losses in tumor karyotypes, our findings suggest that the number of genes that can protect the organism against tumor development may have been underestimated. A multifactorial system seems to be unfolding, reminiscent of other multigenic surveillance systems.

We now postulate that the known tumor suppressor and antiapoptotic genes may be merely 'skyskrapers' in a much vaster landscape. The control of tissue integrity is vitally important. Most vitally important mechanisms are polygenically controlled. While the best known suppressor genes influence the cell cycle and/or apoptosis at the level of the tumor cell precursor, multiple and as yet unknown genetic systems may safeguard tissue integrity. The existence of tumor antagonizing genes, detected in the earlier somatic hybrid studies, and in the present elimination test (see the home page of Stefan Imreh and of Anna Szeles) exemplify, as already indicated, that some genes may antagonize tumor growth in vivo, but not tumor cell growth in vitro. We have given such genes the provisional designation asymmetric tumor suppressor. At present, the list contains the following genes: LTF, L1MD1, HYAL1, HYAL2 and VHL. The inhibition of in vivo growth, without any effect on in vitro proliferation was also confirmed in the gene inactivation test of Eugen Zabarovsky et al. using tet inducible vectors.

Currently, we are looking for additional 'asymmetric suppressors'. We are also exploring the possible relationship of some of these genes to what I have termed 'intercellular surveillance' under Ad.1, section iv above.

We have proposed previously Klein, G. Are there uncharted regions of tumor suppressor genes? IUBMB Life, 2001, 51: 1-3) that genes of the latter type may work in one of the following ways:

i). They might inhibit the the vascularization of the graft;

ii). They may prompt the tumor cell to differentiate in the in vivo microenvironment, but not in vitro;

iii). They may create a more restrictive tissue microenvironment.

We are pursuing these alternatives in a variety of functional studies, partly in collaboration with other groups.