Converting a Normal Cell to a Cancer Cell: A New Tool, a New Insight
October 1, 1999 5 minutes read
Converting a Normal Cell to a Cancer Cell: A New Tool, a New Insight
By David A. Corral, MD
Recently, there has been a significant accumulation in data regarding the biologic processes involved in malignant transformation (i.e., the conversion from a normal phenotype in which cell death is inevitable to a malignant phenotype in which the cell is both immortalized and tumorigenic). While several proteins involved in various pathways, such as the retinoblastoma gene product and p53, have been implicated in tumor development and progression, none has been shown to be solely responsible. In order to produce a human tumor cell from normal cells for in vitro studies, cancer researchers have relied on the use of chemical or physical carcinogens, transfection with an entire viral genome, or painstaking selective culture of malignant cells obtained from surgical or biopsy specimens. A simplified approach to the conversion of normal cells to cancer cells would not only provide a useful tool for in vitro research, but would also shed light on the most elementary essential mechanisms involved in cancer formation. A key question in tumor biology that has emerged over the past decade regards the minimum number of genetic alterations required for malignant transformation.
It has been shown that normal rodent cells can be converted to cancer cells through the co-expression of a minimum of two cooperating oncogenes.1,2 Attempts at duplicating these results in human cells, however, have only met with failure, and the search for the mechanistic differences in cellular control between humans and rodents that accounts for this finding has pointed toward telomere biology for answers.3 Recently, Hahn and colleagues in the laboratory of Dr. Robert Weinberg at the Massachusetts Institute of Technology have demonstrated that ectopic expression of the catalytic subunit of telomerase (hTERT) is the third minimal essential factor necessary for malignant transformation of human cells.4
Telomerase: Of Mice and Men
Normal non-cancerous cells will pass through some seemingly predetermined number of cell divisions before reaching a non-dividing state known as senescence. After some also seemingly predetermined length of time, the cell reaches a "crisis" which is followed by cell death. As the cell passes through its series of successive divisions, the telomeres’ specialized lengths of DNA at the ends of each chromosome become progressively shorter until the "crisis phase" is reached. Thus, it is the erosion of the telomere length which limits the cell’s life span in the normal state. However, cancer cells possess a means of avoiding the crisis phase through the action of telomerase, an enzyme that adds short stabilizing segments of DNA to the telomeres. (See Dr. Kang’s article, Telomerase Activity in Prostatic and Renal Cell Carcinoma, in this issue on p. 54.) Upregulation of telomerase activity in human cancer cells allows the immortalized cell to bypass the normal aging process that concludes with the crisis phase and subsequent cell death. It has been shown in vitro that ectopic expression of the hTERT gene encoding telomerase’s catalytic subunit will enable some, but not all, pre-senescent primary (non-malignant) human cells to multiply indefinitely.4 Thus, it appears that telomerase is able to confer replicative immortality to certain cell types. However, replicative immortality, or "immortalization," is not the only quality necessary for a complete malignant phenotype. The cancer cells must also be able to proliferate and form tumors in an unrestricted manner. Certainly, other oncogenes must be involved to accomplish the total transformation.
Are mice and men created equal? Not in terms of telomeres and telomerase. As it turns out, not only are telomeres inheritantly longer in rodents, but telomerase activity is also constitutively higher. This difference can very well have accounted for the contrasting ability to produce malignant rodent cells through the manipulation of only two oncogenes, vs. the inability to do the same in human cells. Hahn et al threw hTERT expression into the mix of "cooperating oncogenes" to transform human cells.4
Three Necessary Events
The study by Hahn et al identifies a minimum of three genetic events affecting a total of four cellular pathways that result in the conversion of a normal human cell into a tumor cell. These events are ectopic expression of hTERT, the ras oncogene, and the simian virus 40 (SV40) large-T oncoprotein. The authors in the study demonstrated these effects by serially introducing these genes using retroviruses in both human embryonic kidney (HEK) cells and normal human fibroblasts.
Immortalization of HEK cells and fibroblasts was accomplished by the overexpression of the gene hTERT, which encodes the catalytic subunit of telomerase, thereby avoiding the crisis phase in these cells by preventing telomere erosion that would ultimately limit the cell’s life. The ras oncogene is known to be mutated in several human tumors and its expression has been demonstrated to allow cells to grow indefinitely in the absence of growth factors. Introduction of the ras oncogene (H-RAS V12) into HEK cells and fibroblasts resulted in cell senescence which was not overcome by the expression of hTERT. However, when the authors also introduced the oncoprotein SV40 large-T antigen, which transforms normal cells into tumor cells by inhibiting both p53 and retinoblastoma tumor suppressor proteins, the result was increased cell growth. One hallmark of a cancer cell is its ability to form colonies in soft agar (i.e., anchorage-independent growth). Efficient colony formation in soft agar occurred in both HEK cells and fibroblasts only when the combination of all three genes was expressed. Similarly, only cells expressing the combination of hTERT, ras, and large-T demonstrated efficient tumor formation and rapid growth following subcutaneous injection in immunodeficient nude mice. Hahn et al concluded that this combination of three "cooperating oncogenes" that affect a total of four cellular pathways are the minimum genetic events required for malignant transformation.
The work by Hahn et al is clearly a landmark development in the understanding of tumor biology. This study both provides us with the first simple recipe for developing malignant cells in vitro, which will no doubt prove useful in the study of various types of malignancy, as well as multiple aspects of cancer biology. Furthermore, Hahn et al have solidified our concepts of "cooperating oncogenes" in malignant transformation. Elucidation of the mechanisms involved in malignant transformation, however, addresses only the earliest phase of tumor formation. The total effect of cancer on its host is mediated also by the downstream events of angiogenesis, motility and invasion, embolization and circulation, arrest in the capillary beds, adherence to the endothelial walls, extravazation into an organ’s parenchyma, response to the microenvironment, and further tumor cell proliferation and angiogenesis.5 By providing insight into the earliest essential events in cancer formation, the work of Hahn et al and the subsequent models of tumorigenesis of various types of cancer that are likely to develop as a result of this work, will hopefully yield information relevant to the development of cancer prevention strategies as well as identify mechanisms as targets of new therapeutic approaches.
References
1. Land H, Parada LF, Weinberg RA. Nature 1983;304: 596-602.
2. Ruley H. Nature 1983;304:602-606.
3. Weitzman JB, Yaniv M. Nature 1999;400:401-402.
4. Hahn WC, Counter CM, Lundberg AS, et al. Nature 1999;400:464-468.
5. Fidler IJ. Molecular Biology of Cancer: Invasion and Metastasis. DeVita VT Jr., ed. In: Cancer: Principles and Practice of Oncology, 5th Ed. Philadelphia: Lippincott-Raven Publishers; 1997.
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