Changes in the Programmed Cell Death of Polymorphonuclear Granulocytes With Aging Under Various Stimulations

Abstract

Multicellular tumor spharoids (MCTS) are a widely used three-dimensional cell culture system with many advantages compared to monolayer cultures. The differentiation of tumor cells cultured as MCTS resembles more closely the in vivo-situation which is particularly true for their inner, so called quiescent region where tumor cells do not proliferate (leading to a differentiation and proliferation gradient). Most but not all MCTS develop a zone of central cell death until they have an appropriate size. Although this cell death was thought to be necrotic in nature due to insuficient oxygenation or nutrition it has been shown that the cells die at oxygen tensions which allow normal cell growth in monolayer culture. Therefore, we were looking if cells in MCTS die by apoptosis. In three colorectal adenocarcinoma cell lines (HRT-18, HT-29, CX-2) we could show by morphology, TUNEL-method and DNA-laddering that apoptosis in the quiescent area coincides the development of cell death in the center of the MCTS. Supposing that a putative CD95 ligand Will be produced in the quiescent area we studied the FadAF'O-l pathway. Two cell lines, HRT-18 and CX-2, expressed CD95 to the same degree. However, their sensitivities to anti-CD95 mediated apoptosis were different. Because HT-29 did not express CD95, other mechanisms must be active. Heat shock proteins (HSPs) are expressed in most tumor cells at high levels and are known to be regulated also by oxygen tension. It has been shown that members of the HSP70 family and some of the small HSPs have anti-apoptotic properties. Experiments with anti-sense mRNA against HSP7O done by WE1 et al. (Cancer Immunol lmmunother 40, 1995:73-8) revealed that the loss of HSP induce apoptosis.Surprisingly, we found that tumor cells in the quiescent region of MCTS loss all HSPs. These findings suggest that the loss of HSPs my be the initiator of apoptosis found in the quiescent area of MCTS. The regulation of HSPs during hypoxia and their relationship to apoptosis in tumor cells are currently under investigastion.It is wdl known that apoptosis is playing M important role in several disaws such as C(UKXT. In aging mrny alterations are ocmingat oelkrlarand organic M s . Our aim was to study the apoptosis of polymorphonuckar granulocytes (PMNLs), as a might play a role in several pathologies encountmcl with aging. houn of staik culture without ur with stidation. The agents used were: PMA, H202, FMLP, G-CSF, GM-CSF, GSH, LPS, modd,in&wdasevaal-. astheclpoptosis The PMNLa of herhhy young (20-2Syr~), middle (35-5oyrS) and elderly (65-~yn) W= studied for clpoptosiS &er 18IL2. The apoptosis was d by t a d stainiq ofthe platcsandbytlow cytometric mining. It was f d that witbout stimulrtion the susaptibility of PMNb to clpoptosis was slightlyincmscd with aging. Under various stimrlrrtions such as PMA, H& the apoptosk was almost IW?, the FMLP i f l c l w d also, white the other StimULnts d pment to diti;aenb extart the apoptosis. Marked age-rt!htai chngcswae obsesved in the extent of apoptosis under stimulatio...