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the lymphocytic cell line A301 and its infected clone ACH-2 mounted the same
amount of plasma membrane CD95. In contrast, HIV-infected U1 cells had a
consistent decrease in membrane CD95 expression compared with the parental
monocytic line U937. Using an original approach based upon competitive-
quantitative (QC) polymerase chair reaction, we then studied the mRNA levels
of CD95 ligand, CD95L, and we could observe that ACH-2 expressed much
less CD95L mRNA than the parental cell line A301, while in both U937 and
U1 CD95L mRNA levels were below the limit of detection, which was on the
order of 2000 copies/RNA per million cells ( Pinti et al., 1999). To further in-
vestigate the functionality of CD95/CD95L system, we quanti®ed the expres-
sion of di¨erent mRNAs for the total or the membrane form of CD95, the
modulation of such mRNAs, the protein expression on the cell membrane, and
the capacity of infected cell lines of di¨erent origin to trigger apoptosis after
activation of CD95 pathways, as well as under a variety of conditions (Pinti
et al., 2000). We found that infected cells of monocytic origin preferentially
produce the ``protective'' (soluble) form of CD95, and not any detectable
CD95L mRNA, whereas lymphoid cells produce more mRNA for the mem-
brane form of CD95 (which triggers apoptosis) along with reduced but detect-
able amounts of CD95L mRNA. It is possible to hypothesize that, as far as the
CD95/CD95L system is concerned, a complex balance exists between proa-
poptotic events, probably triggered by the host to limit viral production, and
antiapoptotic events, probably triggered by the virus to increase its production
and survival. In cells of monocytic origin, which act as reservoir for the virus,
the antiapoptotic molecules are favored; in cells of lymphocytic origin, mole-
cules with an apoptotic meaning are prevalent.
Further, we performed functional studies and analyzed the capability of one
of these cell lines, U1, to display mitochondrial damages and undergo apoptosis
after stimulation through a classical pathway utilized in human cells of mono-
cytic origin, that of TNF-a. Here we show that, as illustrated in Figure 13.4, U1
cells have a marked decrease in sensitivity to that apoptotic pathway. Indeed,
staining with JC-1 showed that after 6 h of treatment with TNF-a, the per-
centage of cells with relevant mitochondrial depolarization was much lower
than that of the parental cell line, U937. This was con®rmed in parallel samples
by the analysis of samples stained with MitoTracker Red and annexin V ( Fig.
13.5), or with PI (Fig. 13.6). In this case, too, U1 cells showed less mitochon-
drial damage or apoptosis than the parental line U937 (not shown).
FUTURE DIRECTIONS: CYTOFLUORIMETRIC ANALYSIS OF
INTRAMITOCHONDRIAL CARDIOLIPIN DISTRIBUTION
Recent studies have suggested that cardiolipin (CL), a diacidic phospholipid
with an unusually high content of linoleic acid ester residues that is isolated
from beef heart (Hatch, 1996), could be involved in apoptotic cell death (Lieser
et al., 1998; Ushmorov et al., 1999). CL is present throughout eukaryotes, in-
