Spontaneous apoptosis of thymocytes is uncoupled with progression through the cell cycle.
Most developing lymphocytes spontaneously die in the thymus during positive and negative selection of the T cell repertoire. By evaluating the expression of the proliferation antigens Ki-67 and PCNA, we demonstrated here that more than 95% of thymocytes are potentially proliferating. The coincidence within the same cell population of death and proliferation is thus apparent in developing thymocytes. Using dual-parameter cytometric techniques to evaluate in single cells the amount of DNA versus light-scattering values, we found that spontaneous thymocyte apoptosis occurs with similar frequency in all the cycle phases, whereas apoptosis induced by the anti-topoisomerase-II, etoposide (which is the consequence of irreversible DNA damage), takes place with higher frequency in S and G2 phases (i.e., in those cycle phases in which DNA is subjected to torsional constraints). The capability of thymocytes to enter apoptosis was also monitored by digesting DNA in situ with DNase I (a nuclease that cleaves DNA mimicking the nuclear damage common to most apoptotic suicides). We also show that endonuclease-mediated DNA digestion occurs to a similar extent in cells with different DNA contents, i.e., in cycle phases in which the superstructural organization of chromatin is markedly different.
Pellicciari
C
C
Dipartimento di Biologia Animale and Centro di Studio per l'Istochimica del CNR, Piazza Botta 10, Pavia, I-27100, Italy. pelli@ipv36.unipv.it
Bottone
M G
MG
Schaack
V
V
Barni
S
S
Manfredi
A A
AA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D002453:Cell Cycle / Q000276:immunology / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D066298:In Vitro Techniques; D019394:Ki-67 Antigen / Q000378:metabolism; D008854:Microscopy, Electron; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051381:Rats; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D059005:Topoisomerase II Inhibitors
D004791:Enzyme Inhibitors; D019394:Ki-67 Antigen; D018809:Proliferating Cell Nuclear Antigen; D059005:Topoisomerase II Inhibitors; D005047:Etoposide; D004247:DNA