| Summary: | Thesis (PhD)--Stellenbosch University, 2003. === ENGLISH ABSTRACT: The "classic" prostate cell lines, DU145, PC-3 and LNCaP, have served as a valuable
cell biological model for research into prostate cancer. However, their relevance may
be limited because they derive from metastatic, and not from primary normal and
tumour epithelium. The cell lines (1532T, 1535T, 1542T, 1542N and BPH-l) have
been derived from primary benign and malignant human tumour prostate epithelium
and may be more representative. Using these cell lines I have examined the role of
basic cell damage responses (repair, checkpoint activation, apoptosis and associated
signalling proteins, and the influence of androgen status) in cell inactivation, and its
relevance to treatment.
Numerous studies have suggested that loss of p53 function leads to resistance to
chemotherapeutic agents and irradiation. It is shown here that the p53-inactive cell
lines are, in fact, the most sensitive to chemotherapeutic agents such as etoposide,
vinblastine and estramustine, whilst the p53 wild-type cell line, LNCaP, is the most
radiosensitive. Notwithstanding the effects of p53 degradation by the HPV -16 E6
viral protein, the results on chemosensitivity raises the possibility that different
chemotherapeutic agents may have different p53-dependent effects in different
tumour cells.
Androgen deprivation is demonstrated to sensitise prostate cancer cells to
chemotherapeutic agents and it is shown that the hormone independent cell lines are
the most chemosensitive. The LNCaP cell line displayed an increased resistance to
apoptosis induced by etoposide and gamma irradiation, suggesting that androgens are
capable of protection against both these DNA damaging agents.
The major factors determining radiosensitivity in human tumour cell lines are known
to be DNA double-strand break (dsb) induction and repair. In the prostate cell lines I
find that cellular radiosensitivity correlates with the number of DNA double-strand
breaks measured within 2 hours of irradiation, and that the more radioresistant cell
lines show better repair competence. Conclusions as to the influence of androgen dependence on radiosensitivity and repair are not possible at this stage since only the
LNCaP cell line was androgen sensitive. The fact that the 2 hour repair period can
separate radiosensitive from radioresistant cells in 2 groups of human tumour cell
lines highlights the role of non-homologous end-joining repair. This has implications
for therapy, and is consistent with the clinical observation that prostate tumours can
be successfully controlled by low dose rate-brachytherapy.
To evaluate the role of apoptosis, cells were exposed to TD50 concentrations of
chemotherapeutic drugs, and 60Co y-irradiation. Apoptosis was found to be low,
overall, and ranged from 0.1% - 12.1%,3.0% - 6.0% and 0.1% - 8.5% for etoposide,
estramustine and vinblastine, respectively. The percentage of cells undergoing druginduced
apoptosis was, on average, higher in the tumour cell lines than in the normal
cell lines. Gamma irradiation-induced apoptosis levels ranged from 1.3% - 7%. The
LNCaP cell line yielded the lowest percentage of apoptotic cells after exposure. The
l532T cell line yielded the highest percentage of apoptotic cells after exposure.
Apoptotic propensity did not rank the cell lines according to their radiosensitivity.
Immunoblotting demonstrated that the apoptosis-associated proteins, bax and bcl-2,
are expressed at a basal level in all the cell lines tested, but no increase was detected
after exposure to TD50 doses of etoposide, vinblastine and estramustine. The ratio of
bax and bcl-2 also was not altered by DNA damage.
No evidence was found that a correlation may exist between reproductive cell death
and the expression of genes which control apoptosis. My results show that apoptosis
is not a major mechanism of drug- or radiation-induced cell death in prostate cell
lines.
In conclusion, loss of p53 function and loss of androgen dependence was not found to
be correlated with resistance of tumours to chemotherapeutic drugs. Cellular
radiosensitivity was found to be correlated with the number of DNA double-strand
breaks remaining after 2 hours of repair. The more radioresistant cell lines showed
better repair competence. Apoptosis and genes affecting apoptosis, such as p53 and
members of the bcl-2 family, do not seem to contribute significantly to the sensitivity
of prostate cancer cells to anticancer drugs and irradiation. === AFRIKAANSE OPSOMMING: Die klassieke prostaat sellyne, DU145, PC-3 en LNCaP, het 'n waardevolle bydrae
gemaak in die sel biologiese model in prostaat kanker. Die toepaslikheid daarvan
mag egter beperk wees, aangesien hierdie sellyne afkomstig is van metastatiese, en
nie van primêr normale en tumor epiteel nie. Die sellyne 1532T, 1535T, 1542T,
1542N en BPH-I is afkomstig van primêre benigne en maligne menslike prostaat
tumor epiteel en mag moontlik meer verteenwoordigend wees. Deur van hierdie
sellyne gebruik te maak, is die rolondersoek van die reaksie op basiese selskade
(d.w.s. herstel, beheerpunt aktivering, apoptose en verwante sein proteïene, en die
invloed van androgeen status) tydens die proses van sel inaktivering, asook die
toepaslikheid ten opsigte van behandeling.
Volgens verskeie studies lei die verlies aan p53 funksie tot weerstandigheid teen
chemoterapeutiese middels en bestraling. Die resultate van hierdie studie toon dat die
p53-onaktiewe sellyne egter die sensitiefste is vir chemoterapeutiese middels, soos
etoposied, vinblastien en estramustien, terwyl die p53 natuurlike-tipe sellyn, LNCaP,
die meeste radiosensitief is. Ten spyte van die invloed van p53 afbraak deur die
HPV -16 E6 virale proteïen, dui die resultate van chemosensitiwiteit op die
moontlikheid dat verskillende chemoterapeutiese middels verskillende p53-afhanklike
effekte op verskillende tumorselle mag hê.
Dit is bewys dat onttrekking van androgeen prostaat kankerselle sensitiseer teen
chemoterapeutiese middels en dat hormoon-onafhanklike sellyne die hoogste
chemosensitiwiteit vertoon. Die LNCaP sellyn vertoon 'n verhoogde weerstandigheid
teen apoptose wat deur etoposied en y-bestraling geïnduseer is, wat 'n aanduiding is
dat androgene beskerming kan bied teen beide hierdie DNA beskadigingsfaktore.
Die belangrikste faktore wat die radiosensitiwiteit in menslike tumorselle bepaal, IS
bekend dat dit die dubbelbande van DNA verbreek en herstel. Hierdie studie het
aangetoon dat in prostaat sellyne die sellulêre radiosensitiwiteit korreleer met die
aantal DNA dubbelband verbrekings binne 2 uur na bestraling, en dat die meer
radioweerstandige sellyne beter herstelvermoë vertoon. Gevolgtrekkings oor die invloed van androgeen se afhanklikheid van radiosensitiwiteit en herstel kan egter nie
op hierdie stadium gemaak word nie, aangesien slegs die LNCaP sellyn androgeenafhanklik
was. Die feit dat die 2 uur herstelperiode 'n skeiding kan maak tussen
radiosensitiewe en radioweerstandige selle in twee groepe menslike tumor sellyne,
onderstreep die rol van herstel van nie-homoloë endverbindings. Dit hou implikasies
in vir terapie, en stem ooreen met die kliniese waarnemings dat prostaat tumore
suksesvol gekontroleer kan word deur lae intensiteit dosis bragiterapie.
Ten einde die rol van apoptose te ondersoek, is selle blootgestel aan TD50
konsentrasies chemoterapeutiese middels, asook 60Co y-bestraling. Apoptose was oor
die algemeen laag, en het gestrek van 0.1% tot 12.1%,3.0% tot 6.0% en 0.1% tot
8.5% vir etoposied, estramustien en vinblastien onderskeidelik. Die persentasie selle
wat middel geïnduseerde apoptose ondergaan het, was gemiddeld hoër in tumor
sellyne as in normale sellyne. Die waardes van apoptose geïnduseer deur y-bestraling
het gewissel van 1.3% tot 7.0%. Die LNCaP sellyn het die laagste persentasie
apoptotiese selle na bestraling gelewer, terwyl die 1532 r sellyn die hoogste
persentasie gelewer het. Die volgorde van die radiosensitiwiteit van die sellyne was
nie waarneembaar in hulle geneigdheid tot apoptose nie. Immunoblots het aangetoon
dat die apoptose-geassosieerde proteïene, bax en bcl-2, uitgeskei word teen 'n
basisvlak in al die sellyne wat getoets is, maar dat geen verhoogde uitskeiding
waarneembaar was na blootstelling aan TD50 dosisse etoposied, vinblastien en
estramustien nie. Die verhouding van bax en bcl-2 is ook nie beïnvloed deur DNA
beskadiging nie.
Dit blyk daarom dus onwaarskynlik dat daar 'n korrelasie bestaan tussen
reproduktiewe seldood en die uitskeiding van gene wat apoptose beheer. Die resultate
dui daarop dat apoptose me 'n belangrike meganisme vir middel- of
bestralingsgeïnduseerde seldood in prostaat sellyne is nie.
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