Biology Reference
In-Depth Information
the DNA appeared in the hypodiploid peak (Rebeiz et al.
2001
). The G0/G1 peak
decreased to 9
0.5 % (p
0.05) and the G2/M peak became virtually
<
non-existent (0
0.1 %). A significant majority of the cells appeared to be arrested
in early S phase (88
0.05), a phenomenon often observed in cell
cycle dependent apoptosis (Zhu and Anasetti
1995
). Neither ALA alone or Proto
induced apoptosis, nor did these reagents significantly alter the cell cycle when
compared to medium-treated cells (Rebeiz et al.
2001
).
9.8 %, p
<
19.4.5 Abrogation of Induced Apoptosis
by Cycloheximide (Rebeiz et al.
2001
)
The classical inductive pathway of apoptosis, as observed in thymocytes during
negative selection or following treatment with dexamethasone, is defined by
the ability of cycloheximide to inhibit its induction (Cohen et al.
1992
). To deter-
mine whether Oph causes apoptosis via an inductive pathway, MLA 144 cells were
incubated for 18 h with cycloheximide (1.5
g/ml) and then treated for 3 h with
varying amounts of Oph in the continuing presence of cycloheximide. Cells were
then double-labeled (Cohen et al.
1992
) with Hoechst 33342 and PI. In flow
cytometric analysis, apoptotic cells are represented by high Hoechst 33342 and
low forward angle light scatter (FALS) with the PI positive (necrotic) cells excluded.
Oph at concentrations ranging from 0.375 to 1.5 mM induced a significant increase
in the proportion of apoptotic cells, with the highest level of apoptosis occurring at
0.75 mM (85 %, p
μ
0.05). Medium-treated cells, which were serum-deprived for
3 h, were 5 % apoptotic. Cycloheximide alone increased the apoptotic population in
medium-treated cells to 15 %, however this was not statistically significant.
Co-incubation of cells with cycloheximide and Oph decreased Oph-induced apo-
ptosis. The percentage of apoptotic cells was reduced to background levels at all
doses of Oph (Rebeiz et al.
2001
). These results indicated that Oph causes apoptosis
via the classic induction pathway that requires protein synthesis.
<
19.5
Induction of Tumor Necrosis by ALA
and Oph-Dependent Photodynamic Therapy
As was mentioned previously the approved use of ALA and its long chain fatty acid
esters to treat skin cancer has met with limited acceptance by the medical commu-
nity, because of damage to surrounding tissues and limited effectiveness as com-
pared to liquid nitrogen treatments (oral communication from my skin doctor).
In this section we will describe, as an alternative, the use of AlA and Oph as a
model research system for treating Meth-A cancerous cells and solid tumors.
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