Normal human somatic cells in culture have a limited dividing potential. This is due to DNA end replication problem, whereby telomeres shorten with each subsequent cell division. When a critical telomere length is reached cells enter senescence. To overcome this problem, immortal HeLa cell line express telomerase, an enzyme that prevents telomere shortening. Although immortal, the existence of non-dividing cells that do not incorporate (3)H-thymidine over 24 h of growth has been well documented in this cell line. Using DiI labeling and high-speed cell sorting, we have separated and analyzed fractions of HeLa cells that divided vigorously as well as those that cease divisions over several days in culture. We also analyzed telomerase activity in separated fractions and surprisingly, found that the fraction of cells that divided 0-1 time over 6 days in culture have several times higher endogenous telomerase activity than the fastest dividing fraction. Additionally, the non-growing fraction regains an overall high labeling index and low SA-beta-Gal activity when subcultured again. This phenomenon should be considered if telomerase inhibition is to be used as an approach to cancer therapy. In this paper we also discuss possible molecular mechanisms that underlie the observed results.

Normal human somatic cells have a limited division potential when they grow in vitro. It is believed that shortening of telomeres, specialized structures at the ends of chromosomes, controls cell growth. When one telomere achieves a critical minimal length, the cell cycle control mechanism recognizes it as DNA damage and causes the cell's exit from the cycle in G1-phase. Because it is not possible to extend telomeres in normal cells, this non-dividing state is prolonged indefinitely, and is known as cellular senescence. The immortal cell line MDA-MB-231 has active telomerase, which prevents telomere shortening and allows cells' permanent divisions. However, there is a fraction of cells that do not divide over several days in culture as documented for some other tumour cell lines. Combination of methods has made it possible to isolate these non-growing cells and compare them with the fraction of fast-growing cells from the same culture. Although the non-growing fraction contains a significant percentage of typical senescent cells, both fractions have equal telomerase activity and telomere length. In this paper we discuss possible mechanisms that cause the appearance of this non-growing fraction of cells in cultures of MDA-MB-231, which indicate stress and genome instability rather than variation in telomerase activity or telomere shortening to affect individual cells.

The RecA loading activity of the RecBCD enzyme, together with its helicase and 5′ → 3′ exonuclease activities, is essential for recombination in Escherichia coli. One particular mutant in the nuclease catalytic center of RecB, i.e., recB1080, produces an enzyme that does not have nuclease activity and is unable to load RecA protein onto single-stranded DNA. There are, however, previously published contradictory data on the recombination proficiency of this mutant. In a recF– background the recB1080 mutant is recombination deficient, whereas in a recF+ genetic background it is recombination proficient. A possible explanation for these contrasting phenotypes may be that the RecFOR system promotes RecA-single-strand DNA filament formation and replaces the RecA loading defect of the RecB1080CD enzyme. We tested this hypothesis by using three in vivo assays. We compared the recombination proficiencies of recB1080, recO, recR, and recF single mutants and recB1080 recO, recB1080 recR, and recB1080 recF double mutants. We show that RecFOR functions rescue the repair and recombination deficiency of the recB1080 mutant and that RecA loading is independent of RecFOR in the recB1080 recD double mutant where this activity is provided by the RecB1080C(D–) enzyme. According to our results as well as previous data, three essential activities for the initiation of recombination in the recB1080 mutant are provided by different proteins, i.e., helicase activity by RecB1080CD, 5′ → 3′ exonuclease by RecJ- and RecA-single-stranded DNA filament formation by RecFOR.