Biology Reference
In-Depth Information
19.2.1
Identification of the Porphyrin That Accumulated
in MLA 144 Cells After Treatment
with
Aminolevulinic Acid and
1,10-Phenanthroline as Protoporphyrin IX
δ
Since herbicide and insecticide investigations revealed that Oph induced tetrapyrrole
that it may do the same in cancer cells.
Therefore Initial investigations attempted to determine whether significant
Proto biosynthesis and accumulation could be induced in proliferating MLA
144 gibbon lymphoma cells after incubation in total darkness with 1 mM ALA
and 0.75 mMOph. It was observed that The ALA + Oph treated cells accumulated
large amounts of a pigment that exhibited emission and Soret excitation maxima
at 298 K and 77 K identical to authentic Proto (Rebeiz et al.
1992
). After Mg
insertion, the pigment exhibited identical fluorescence properties as Mg-Proto
(Rebeiz et al.
1992
).
To further confirm the identity of the accumulated product, its HPLC mobility
was compared to authentic Proto on a Waters Bondapack reverse phase C18-bonded
column. Authentic Proto, dissolved in methanol, and the pigment extracted from
MLA 144 cells exhibited similar retention times of 5.4-5.6 min (Rebeiz et al.
1992
).
Since the fluorescence properties of this pigment at 298 K and 77 K were identical to
authentic Proto before and after Mg insertion, and since its HPLC mobility was
similar to that of authentic Proto, the observed fluorescence was ascribed to the
biosynthesis and accumulation of Proto by the treated cells. Protoporphyrin IX
accumulation in the treated cells amounted to 29.4 nmol/100 mg of cell protein
(Rebeiz et al.
1992
). After the same period of dark-incubation Proto was not detected
in untreated cells. It was therefore concluded that after treatment with ALA and Oph,
MLA 144 cells accumulated large amounts of Proto.
19.2.2
Induction of Cell Lysis of MLA 144 Cells
Treated with ALA and Oph
Next, the extent of cell destruction in the light following the induction of tetra-
pyrrole accumulation by MLA 144 cells was investigated. The cells were labeled
with Na
51
CrandtheninducedtoaccumulateProtobytreatmentwith1mMALA
and 0.75 mM Oph for 3.0 h in darkness. After exposure to white light (sodium
halide, 2.11 mW cm
2
) for an additional 30 min, cell destruction was evaluated
by monitoring the release of
51
Cr into the incubation medium. ALA + Oph-
treated cells that had accumulated Proto exhibited severe photodynamic damage
in the light as compared to control cells which had not accumulated any
tetrapyrroles (Rebeiz et al.
1992
).
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