The Immortalization Phenomenon

Abstract

Mouse embryonic fibroblasts cultured at 21% O₂ spontaneously immortalize, escaping the replicative limit by a route presumed telomere-independent (p53/p19ARF loss plus telomerase or ALT activation). This paper raises an untested question: what happens to the centriole when a cell line immortalizes? If the centriole is a physical counter of replicative age, its accumulated damage should survive the event that erases the Hayflick limit — a persistent counter carried into the immortal future; if it is reset during immortalization, it is not a counter. We propose a pre-registered, three-arm experiment that (i) reproduces the immortalization phenomenon in MEFs at 21% vs 3% O₂, (ii) measures the clean telomere-driven limit in human fibroblasts at 3% O₂, and (iii) tracks centriolar damage markers (scaffold geometry, subdistal appendages, PTM load, chemical damage) across 20 and 50 post-escape passages, discriminating passive dilution from active reset through degradation inhibitors and isotope tracing, and separating the counter from the damage that reports it. The decisive sub-experiment sorts cells by centriole generational depth before the crisis and counts divisions per subpopulation via ARGUS lineage tracking. Persistence of pre-crisis damage in the immortalized line would demonstrate that a cellular structure carries replicative history through the very event that abolishes the Hayflick limit — direct evidence for a physical, non-dilutable centriolar counter.