A dissertation submitted to the Faculty of Science
University of the Witwatersrand, Johannesburg
for the Degree of Masters of Science.
Johannesburg 1995 / This study ~valuates the expression ofthe m1U\lAand protein products flfthebClw
2 prato-onCPllenein the.b1.lman myelooytlc THP~land.Hl',."60cell lines. The bcl..2
Pl'oto-llcogerle codes for a protein termerJ. nCL-2. that is able. to inhibit sO~1'le
pa~hways tWprogrammed celt death, Both c;~lFlines are. capable of und~rgomg
programmed cell death, as. is shown by the presence of some apoptosing cells in
cell morphQlogy studies after 24 11our·stimulations with the protein synthesis
inhibitor pummycin(1 n.g/ml). Some HL~60 cells, but no TlIP ..I. cells, undergo
apo!ptosis upon 24 pout co-stinrulation with the phorbol ester PMA (16 nMrand
the PKA activator dbcAMP (50 ~M). Stimulating with PMl\ or dhcAM.P alone
.,
does not induce detectable levels of. apoptosls in THP ..1 and lIL-60 cells.
Bt.L-2 expression is inversely related to differentiation in myeloid cells, and
mature tnonoc}'tes express no :aCt..2. Cell cycle analyses and [3HJthymidine
uptake studies show that PMA (16 nM) decreases proliferation both in THP..1
cells and the less differentiated HL~60 cells, While dbcAMP (50. ",M) decreases
proliferation markedlyin TI:W ...1 cells only e . Cell adhesion and cell morphology
studies show that PMA, but not abeAM?, induces differentiation in both cell
lines.
Sthnulating cells witli PMA for 24 hours induces the expression Of IL-IB mRNA
and protein in THP ..1 cells, but. not in the less differentiatt:.d· BLooO cells,
~ldicatingthat n.,.·Hl expression. is .related to differentiation itr~~~e .celllines.
The expression of bc1-2 mRNA and BCL~21)r()tein ia.analyseQr'ant:1. compared itt .
both •cell lines, both undet undifferentiated cQnditittms' andiafter induction,.of
idlffere;nl;iation. bel-?, mRNA expression is eval~lated using reVCJtselransciption and
polymerase. chain reaction amplification memods, and is sho~,.nto decrease upon
PMA:-stimulation in both cell lines. bcl..Z mRNA exptes,~i()n :isshown to decreese
to less than 5 % of contrpl values irt.TB:P~1 cells (n;::;2) and ,to1;)etweert10 % and
20. % of.cohttol values in HL-60 cells (11::;,3) after24 houtitof PlVtA stirnuiatiol1.
be;..2 mRNA expression aite!' dbeAMP sti.umldion may·,d~teaseor may remain
unchanged in both cell lines When C(tmp~rv' ( to unstimulfated cells.
sIotblotting' methods is described qllantitatively. in HL~60 cells, hut only
qua1itativ~ly in Tap..I cells where .low levels. 0'( BCL-2 ••e•xp]('essio~ made
quantit.ation. technically difficult. :aCL~2 eJtpresston is shown to decrease. to 78 %
of control values by 24 hours after PMA stimulation in HLh6() cells. The );}ICL..Z
p.rotl~in"xpresslon is betvlleen 5 X and 15 X lower in THP-l cells than in HL-60
cells. The inverse relationship of myeloid cell differentiation'to bel..2 mRNA and
BCL ..2 pl;otein expression is confirmed by these results. Stimulation with dbcAMP
does not cause changes in BCL-2 protem ,.::xpressi(>n.in '7,.HP-l cells.
Immunocytochemical detection of 'SCL-:2protein in w\v)le cells is describ~. The
protein is shown to be located mtAultly in the cytoplnsm. Stronger positive staining
is observed in HL ..60 cells .than in .THP ..1 cells, thus confirming the results
obtained from protein slotblots.
IThiS:!Rtudy confirms the inverse relationship of myeloid cell differentiation to bcl-2
,,'m, RNA expression and to BCL·2 protein expression. MO!1(lcytes and tis~ue
macrophages .have a high turnover rate' compared ti;) their, ,ha,ematopoietic stem·
cells, and are 'c,msistently rep1aceJ by newly differentiated progeny cells. High
BCL-Z expressi911 in the long.:;1ivedstem cells will help' pro«!ct them/ from cell,
death, jDecreases in PCL~2 expressiQU during differentiation y,villlead to increased
susceptibility to programmed cell death, and will allow for the high turnover rate
observed in Ul0i10Cytes:and macropll~ges. Tissue homeostas~s is maintainoo by this
balance between cell proliferation and cell death.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/20612 |
| Date | 15 July 2016 |
| Creators | Becher, Anja |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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