| Abstract |
Prostate cell lines were derived from two regions of prostate tissue from
the same patient. The objective was to produce cell lines (as a useful in
vitro model) from these two different regions which exhibit different
properties for carcinoma development. The tissue was obtained from
patients suffering from benign prostate hyperplasia undergoing trans-
urethral resection. Tissue was taken from the deep (peripheral) and
superficial (peri-urethral) areas. The cells were immortalised by
transduction with constructs over expressing the cdk4 and hTERT genes.
These cell lines were then characterised for their cellular phenotypes
utilized for radiation transformation studies and utilized to investigate
the role of plant derived polyphenols on normal and tumour cells.
The cell line from the superficial region (P21s) was treated to
fractionated doses of gamma radiation and a transformed cloned cell line
was derived (P21s 40Gy (clone-a)). The cell line from the deep region
(P21d) was found to consist of a mixed population of abnormal cells and a
transformed cloned cell line was derived from it (P21d 40Gy (clone-a). In
an attempt to obtain a normal P21d cell line cloned cell lines from early
passage P21d cells were established. All seven cloned lines were abnormal
with an average of 80 chromosomes per cell, invasive using a Matrigel
assay and produced anchorage independent colonies. All cell lines were
fully characterised with immunocytochemistry, chromosome analysis,
invasion assays, and anchorage independent colony formation. P21s
expressed basal cell markers (cytokeratin 5 (CK5) and 14), were positive
for stem cell markers (prostate specific stem cell antigen PSCA, CK6),
positive for p16, p63 and telomerase expression and negative for c-Myc
expression. P21s was not invasive in a Matrigel assay and did not produce
anchorage independent colony formation. P21d and P21d 40Gy (clone-a) also
expressed CK5, CK14, PSCA, CK6, and telomerase but not p16 or p63 and
showed an increase in expression of nuclear c-Myc, highly invasive and
produced anchorage independent colonies. P21s 40Gy (clone-a) expressed
CK5, CK14, PSCA, CK6, telomerase and p63, produced anchorage independent
colonies, and was weakly positive for c-Myc expression.
Spectral karyotyping analysis (SKY) showed P21s had a normal chromosome
complement except an additional chromosome 20 whereas the P21s 40Gy
(clone-a), P21d and P21d 0Gy (clone-a) cell lines had an abnormal chromosome
complement with P21d and P21d 0Gy (clone-a) cell lines expressing multiple
copies of every chromosome including loss of the Y chromosome. These
results were echoed in the single nucleotide polymorphism chip (SNP)
results which showed P21s as normal but P21d and P21d 40Gy (clone-a) to
have large deletion and amplification regions that correlated with the SKY
analysis.
No differential cytotoxic response was noted between normal and abnormal
cell lines including prostatic carcinoma cell lines LNCaP and PC-3
following treatment with strawberry polyphenol compounds. Most reports of
a cytotoxic response to tumour cells in the literature did not compare the
response to normal cells and used established cell lines. Human
lymphocytes were also tested and all compounds were toxic in high doses.
Polyphenol and ellagitannin rich polyphenol fractions were very cytotoxic
and the anthocyanin rich fraction less toxic. In contrast to the lack of a
direct differential cytotoxic effect, plant polyphenols did produce a
protective effect to a carcinogenic insult. However a protective effect
was noted via micronucleus assay with 3 hour incubation with the
polyphenol rich fraction prior to radiation treatment. Finally, the
expression and association of metabolic enzymes within the cells cytosol
were investigated. The P21s cells were found to express both isoforms of
LDH and so thought to be able to metabolise anaerobically and aerobically.
P21d and P21d 40Gy (clone-a) cells were found to only express one isoform
in the complex and so it was assumed that these cells favoured anaerobic
metabolism of ATP in correlation to the Warburg effect. c-Myc association
with compounds in the cell cytosol of P21s cells existed whereas, abnormal
cells lost this association along with up-regulation of c-Myc expression
and down stream targets of c-Myc in the nuclei.
Thus these newly established human prostate cell lines provide a useful
model system for investigating the biology of the prostate and prostate
cancer.
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