Immortal ALT⁺ human cells do not require telomerase reverse transcriptase for malignant transformation

Many human cancer cells lack telomerase activity but nevertheless maintain telomeres via a process termed "alternative lengthening of telomeres" (ALT). Despite being immortal and having a telomere maintenance mechanism, ALT⁺ human fibroblasts require telomerase reverse transcriptase (hTERT) for tumor formation in immunodeficient mice when tested by s.c. injection. Here we show that three ALT⁺ human SV40-immortalized fibroblast cell lines require only oncogenic Ras^V12G to be converted to a fully tumorigenic state. When cells were implanted beneath the kidney capsule of immunodeficient mice, they invaded the kidney and neighboring organs and metastasized to the lungs. Ras^V12G-expressing ALT⁺ cells remained completely telomerase negative. Introduction of hTERT conferred strong telomerase activity but did not appreciably change the malignant properties of the cells. However, when cells were tested by s.c. injection, Ras^V12G-transduced ALT⁺ cells did not form tumors, and in this site, hTERT was required for tumorigenicity. These data show that when the s.c. injection method is used as an assay for tumorigenicity, hTERT may be artifactually scored as an oncogene; the subrenal capsule assay shows that ALT, as a telomere maintenance mechanism, is equivalent to hTERT in neoplastic transformation of human cells by oncogenes.