Telomerase and Telomere Dynamic in Normal and Malignant Stem Cells

A genetic change necessary in all successful cancers is the development of immortality. All normal cells, including stem cells, are limited in the number of cell divisions they can undergo (Hayflick limit) and will eventually undergo terminal senescence. In contrast, in acute leukemias (but not in chronic leukemias) cells are immortal and can undergo indefinite replication. The biological clock that limits the replication of normal cells is thought to be a tumor resistance mechanism since cancer begins with the mutation of a single cell and the malignant clone would need to divide 60-80 times to appear as clinically observable disease (1010-1011 total cells). If the biological clock mechanism is intact, premalignant clones would undergo senescence after 30-40 cell divisions and this is probably the fate of the majority of such clones. However, genetic changes can occur that allow cells to escape their biological clock.

The mechanism behind the biological clock is based on the telomeric DNA at the chromosome ends. At each cell division a small amount of telomeric DNA (50-100 base pairs) is lost and after 30-50 cell divisions the cumulative telomeric DNA loss is recognized as “damaged” DNA and molecular pathways are activated that trigger cell senescence or apoptotic death. The enzyme telomerase is a reverse transcriptase capable of preventing telomere shortening and can even restore and elongate eroded telomeric DNA.

The Moore laboratory has published 19 scientific papers showing the importance of telomerase expression in a variety of human cancers, including leukemia. In a recent study we have shown that the level of telomerase expressed in malignant multiple myeloma calls was highly predictive of survival at 4 years with 80% of patients dead with high levels of telomerase while only 40% were dead with lower levels (p=<0.002). Telomerase is the result of expression of two genes-hTR coding for the RNA “template” component and hTERT coding for the protein component of the enzyme. We and others have shown that introduction of the hTERT gene into a variety of normal cells (fibroblast, endothelium, hematopoietic stem/progenitors) results in expression of high levels of active telomerase enzyme associated with protection of telomeres from DNA loss resulting from cell division. In normal cells the addition of hTERT by transduction with retroviral or lentiviral vectors results in immortalization but retention of normal growth control and differentiation i.e. no premalignant changes. If additional genetic changes are introduced such as addition of mutated Ras genes and viral T antigen then hTERT addition results in malignant transformation (e.g with normal breast epithelium or vascular endothelium).

Since telomerase expression is a necessary component of the malignant process it also presents an attractive target for anti-cancer therapy. The Moore laboratory has demonstrated that hematopoietic malignancies (leukemias, lymphomas, multiple myeloma) express telomerase yet have comparatively short telomeres. This makes them potential targets for telomerase inhibition since the short telomeres ensure that there is a rapid onset of senescence/death once telomerase activity is blocked. We have developed an oligonucleotide that acts as a specific telomerase template inhibitor, blocking telomerase activity in vitro and in vivo in human tumors growing in immunodeficient mice. This anti-telomerase therapy was effective in suppressing lymphoma, myeloma and leukemia development in this xenograft model.Telomere shortening within the hematopoietic and immune system has been shown to correlate with aging.

We have studied the impact of extensive chemotherapy and stem cell transplantation on telomere length within the hematopoietic system of cancer patients. In a large study of over 250 patients with Multiple Myeloma, undertaken in collaboration with Dr B. Barlogie of the University of Arkansas Medical center, we have followed telomere kinetics and stem cell function over a five year period of therapy. This data base is providing valuable information that relates to the impact of aging and cancer therapy on stem cell function and telomere kinetics. We have identified a subset of individuals who actually elongated their leukocyte telomeres despite extensive myelosuppressive therapy. The genetic and molecular basis of this is under investigation.

Engelhardt M, MacKenzie K, Drullinsky P, Silver RT, Moore MAS. Telomerase activity and telomere length in acute and chronic leukemia, pre- and post-ex vivo culture. Cancer Res. 2000; 60:610-617.

MacKenzie KL, Franco S, May C, Sadelain M, Moore MAS. Mass cultured human fibroblasts overexpressing hTERT encounter a growth crisis following an extended period of proliferation. Exp Cell Res 2000, 259:336-350.

Franco S, MacKenzie KL, Dias S, Alvarez S, Rafii S, Moore MAS. Clonal variation in phenotype and lifespan of human embryonic fibroblasts (MRC-5) transduced with the catalytic component of telomerase (hTERT). Exp Cell Research, 2001, 268: 14-25.

Boklan J, Nanjangud G, MacKenzie KL, May C, Sadelain M, and Moore MAS. Limited Proliferation and Telomere Dysfunction Following Telomerase Inhibition in Immortal Murine Fibroblasts. Cancer Research 2002; 62:2104-2114.

MacKenzie KL, Franco S, Naiyer AJ, May C, Sadelain M, Rafii S, Moore MAS. Multiple stages of malignant transformation of human endothelial cells modeled by co-expression of telomerase reverse transcriptase, SV40 large T antigen and oncogenic N-ras. Oncogene 2002;21:4200-4211.

Franco S, Ozkaynak MF, Sandoval C, Tugal O, Jayabose S, Engelhardt M, Moore MAS. Telomere dynamics in childhood leukemia and solid tumors: a follow-up study. Leukemia 2003. 17:401-410.

Wu K, Orme L, Shaughnessy J, Jacobson JJ, Barlogie B, Moore MAS. Telomerase and telomere length in multiple myeloma: correlations with disease heterogeneity, cytogenetic status and overall survival. Blood. 2003 101:4982-4989.

Wang ES, Wu K, Chin AC, Pongracz K, Gryaznov S, Moore MAS. Telomerase inhibition with an oligonucleotide telomerase template antagonist: in vitro and in vivo studies in multiple myeloma and lymphoma. Blood 2004;103: 258-66.

Gammaitoni L, Weisel KC, Gunetti M, Wu KD, Bruno S, Pinelli S, Bonati A, Aglietta M, Moore MAS, Piacibello W. Elevated telomerase activity and minimal telomere loss in cord blood long-term cultures with extensive stem cell replication. Blood 2004, 103:4440-8.