Thesis (Ph.D.)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Predictive assays are of key importance in clinical radiotherapy, chemotherapy and toxicology. Prior to exposing malignant tissues to irradiation or drugs in the clinic, a good understanding of the damage response to the cytotoxic agent is required. Such information is necessary for effective planning and treatment. Regrettably however the methods which detect DNA damage, namely micronucleus, apoptosis and DNA repair assays do not rank cells according to their intrinsic survival response to cytotoxic agents. The application of predictive assays based on micronuclei and apoptosis in the clinic therefore remains unreliable. Using a panel of 7 neuroblastoma and 6 neuroepithelial cell lines, it is shown that damage assays also do not rank cell lines according to cell survival. However, radiosensitivity can be reconstructed from micronuclei formation and apoptosis, and a new parameter, cell death due to small deletions, chromosome aberrations and misrepair. The interrelationships between radiation-induced micronuclei, apoptosis and repair is complex and varies between cell lines. Micronuclei formation and apoptosis are exponentially interrelated. This suggests that these cell inactivation pathways are strongly correlated. Evidence exists to show that the expression of apoptosis and micronuclei is influenced by the extent of DNA double-strand break repair within the first 2 hours after irradiation. Cell lines which repair more damage in the first 2 hours express more micronuclei and less apoptosis. Micronuclei formation and apoptosis and are not significantly correlated with the 20 hours slow repair component. There is however a strong correlation between 20 hours of repair and radiosensitivity, with the more radioresistant cell lines being more repair proficient. This suggests that the 2 hours (fast) DNA repair component is more error prone, and that cells lines repairing more damage late after irradiation tend to show better survival. In conclusion, micronuclei formation, apoptosis and DNA repair are strictly cell type specific and are not suitable for predicting radiosensitivity in terms of cell survival. However, these assays are very useful for studies on the influences of dose modifying agents i.e. oxygen tension, radiation modality, pH, cytotoxic sensitisers and radiation protectors which alter cellular responses and provide insight into damage mechanisms. / AFRIKAANSE OPSOMMING: Toetse wat kliniese gevolge kan voorspel is van uiterse beking in stralingsterapie, chemoterapie en toksikologie. Voordat kwaadaardige weefsels aan bestraling of chemise middels blootgestel can word in die kliniek, moet daar 'n goeie begrip van die skade weerstand wees van die selgiftige middel. Hierdie inligting is noodsaaklik vir effektiewe beplanning en behandeling. Ongelukkig stem die metodes wat ONS skade, apoptose en ONS hersteltoetse, nie ooreen met die selle se inherente straling sensitiwiteit nie. Die aanwending van voorspelbare toetse gebaseer op mikrokerne en apoptose in die kliniek bly dus onbetroubaar. Deur gebruik te maak van 'n paneel van 13 neurologiese sellyne, is daar bewys dat ONS skade toetse nie sellyne rangskik volgens seloorlewing nie. Radiosensitiwiteit kan herbou word deur 'n neiging om mikrokerne te vorm, apoptose, en sel sterftes weens klein vermiste ONS volgordes, chromosoom aberrasies en verkeerd herstelde ONS. Die verhouding tussen straling-geïnduseerde mikrokerne, apoptose en selgenees is kompleks en varieer tussen sellyne. Die ontstaan van mikrokerne en apoptose is eksponensiel verbind. Dit dui aan dat hierdie seltraagheidsbane streng gekorreleer word. Daar is bewys dat die uitdrukking van apoptose en mikrokerne deur die mate van herstel van die ONS dubbelstring-breuke binne die eerste 2 ure na bestraling beïnvloed is. Daar is gevind dat sellyne wat meer skade herstel binne die eerste 2 ure meer mikrokerne en minder apoptose toon. Die ontstaan van mikrokerne en apoptose is nie betekenisvol gekorreleer met die 20-uur stadige herstel komponent nie. Daar is inderdaad 'n sterk korrelasie tussen die 20-uur herstel komponent en radiosensitiwiteit, en die meer radioweerstandbiedende sellyne net In hoër herstel bekwaamheid. Dit laat mens dink dat die 2 uur (vinnige) DNS herstel komponent meer geneig is om foutief te wees, en dat sellyne wat meer skade, laat na bestraling herstel, beter oorlewing toon. Ten slotte, die ontstaan van mikrokerne, apoptose en DNS herstel is strenggesproke seltipe spesifiek en is nie toepaslik om radiosensitiviteit, in terme van seloorlewing, te voorspel nie. Hierdie toetse is nuttig vir studies waar die invloed van dosismodifiseringsagente, soos suurstof-spanning, straling-tipe, pH, sitotoksieke sensiteerders en stralingsbeskermers, wat sellulêre gevoeligheid verander en insig gee tot skade meganismes.

Thesis (PhD)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Pentoxifylline enhances the toxicity of radiation and has emerged as an effective modulator of the radiation response of tumour cells. The molecular mechanisms involved in the enhancement of radiotoxicity by pentoxifylline have not yet been elucidated. Cell cycle blocks, DNA repair and programmed cell death (apoptosis) are all pert of the cellular response to DNA damage and as such must be considered as targets of the drug. In this study, the influence of pentoxifylline on radiosensitisation, G2 block abrogation, DNA repair inhibition and the induction of apoptosis have been investigated in 8e11 and MeWo melanoma and 4197 and 4451 squamous cell carcinoma (SCC) cell lines. The influence of pentoxifylline on radiation-induced apoptosis in Jurkat J5 T-lymphocytic leukemia cells has also been assessed. Hela cervical carcinoma cells were used to investigate the molecular events involved in the abrogation of the G2 block by pentoxifylline. It is shown that pentoxifylline preferentially sensitises the TP53 mutant MeWo and 4451 cell lines and enhances radiotoxicity by factors of up to 14.5. In the MeWo melanoma, but not in the 4451 SCC cell line, radiosensitisation is accompanied by inhibition of DNA repair. No significant enhancement of radiation-induced apoptosis was observed in MeWo melanoma and 4451 SCC cells. However, Jurkat J5 cells showed an increase in apoptosis after irradiation in the presence of the drug. In irradiated Hela cervical carcinoma cells, pentoxifylline affects the expression of the two components of the mitosis promoting factor (MPF), namely cyclin 81 and p34cdC2, and rapidly restores cyclin 81/p34cdC2 ratios to control levels. Analysis of cyclin 81 expression in whole cells and isolated nuclei furthermore reveals an influence of the drug on the subcellular translocation of the MPF. It is concluded that G2 block abrogation is not the only mechanism involved in the radiosensitisation of tumour cells by pentoxifylline, but that DNA repair inhibition plays a role in certain cell types. Although pentoxifylline induces apoptosis in Jurkat J5 thymocytes, radiation-induced apoptosis plays no role in the radiosensitisation of the two TP53 mutant melanoma and sec cell lines. Abrogation of the G2 block by pentoxifylline, which sensitises tumour cells to a second irradiation or chemotherapeutic challenge, involves a modulation of the levels of cyclin 81 and p34cdC2, and the subcellular location of the MPF. These results are of utmost importance for the clinical potential of pentoxifylline as a dose modifier in cancer therapy. / AFRIKAANSE OPSOMMING: Pentoxifylline verhoog die toksisiteit van bestraling en het dus na vore getree as 'n effektiewe modulator van die sellulêre stralingsrespons in kankerselle. Die molekulêre meganismes betrokke by die verhoging van stralingstoksisiteit deur pentoxifylline is egter nog nie duidelik nie. Blokkering van die selsiklus, die herstel van ONS skade en geprogrammeerde seldood (apoptose) vorm almal deel van die sellulêre respons ná bestraling en word as sulks beskou as potensiële teikens van die middel. In hierdie studie is die invloed van pentoxifylline op stralings-sensitiwiteit, G2 blok verwydering, die vertraging van ONS herstel en die indusering van apoptose ondersoek in die Be11 en MeWo melanoom en 4197 en 4451 plaveisel-sel karsinoom sellyne. Die invloed van pentoxifylline op stralings-geïnduseerde apoptose in Jurkat J5 T-limfosiete is ook bestudeer. Hela servikale karsinoom selle is gebruik om die molekulêre gebeurtenisse rondom die verwydering van die G2 blok deur pentoxifylline te ondersoek. Dit word aangetoon dat pentoxifylline by voorkeur die radiosensitiwiteit van die TP53 mutante MeWo en 4451 sellyne verhoog, en stralingstoksisiteits verhogingsfaktore van tot 14.5 genereer. Hierdie effek gaan gepaard met die vertraging van ONS herstel in die MeWo melanoom, maar nie in die 4451 plaveisel-sel karsinoom sellyn nie. Die meting van apoptose toon geen noemenswaardige verhoging van stralings-geïnduseerde apoptose in MeWo melanoom óf in 4451 plaveisel-sel karsinoom selle nie. Jurkat J5 T-limfosiete toon egter wel 'n verhoging in apoptose wanneer bestraling in die teenwoordigheid van pentoxifylline gedoen word. In Hela servikale karsinoom selle affekteer pentoxifylline die uitdrukking van die twee komponente van die mitose promoverings faktor (MPF), naamlik siklien B1 en p34CdC2 , en restoreer die siklien 81/p34cdC2 verhoudings vinnig na normale vlakke. Ontleding van die siklien 81 uitdrukking in heel selle en in geïsoleerde selkerne toon verder dat die middelook die sub-sellulêre ligging van die MPF affekteer. Die gevolgtrekking word gemaak dat G2 blok verwydering nie die enigste meganisme is waardeur pentoxifylline radiosensitiwiteit verhoog nie, maar dat die vertraging van ONS herstel in sommige seltipes 'n rol speel. Alhoewel pentoxifylline apoptose verhoog in T-limfosiete, speel dit nie 'n rol in die verhoogde radiotoksisiteit wat waargeneem is in die TP53 mutante melanoom en plaveisel-sel karsinoom sellyne nie. Verwydering van die G2 blok deur pentoxifylline, wat selle meer sensitief kan maak vir 'n tweede stralings- of chemoterapie aanslag, behels die modulasie van siklien 81 en p34cdc2 vlakke en die sub-sellulêre ligging van die MPF. Hierdie resultate is van uiterste belang vir die kliniese aanwending van pentoxifylline as 'n dosis-modifiseerder in kankerterapie.

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.

Thesis (MMed)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Background: HIV is the defining pandemic of our era, with an estimated 5.9 million people infected in South Africa according to World Health Organization (WHO) estimates for 2011. The expression and treatment of non-AIDS defining cancers has become an important consideration in this cohort, as antiretroviral therapy (ART) has prolonged survival in a subgroup previously at risk of early mortality. Study Design: A retrospective cohort study of all women seen at the Combined Breast Cancer Clinic at Tygerberg Hospital between January 2010 and December 2011, stratified into three subgroups based on HIV status. Methods: Of the 816 patients screened, 586 met inclusion criteria; of which 31 (5.3%) patients were HIV positive, 420 (71.7%) HIV negative, and 135 (23%) had an unknown HIV status. The disease phenotype was described in each subgroup, as well as toxicities associated with standard chemotherapy regimens, with an emphasis on completion rates of systemic cytotoxic treatment. The insult of cytotoxic therapy to the CD4 count was described for this cohort. Results: Women with HIV had a statistically significant (p<0.001) younger age at presentation of breast cancer with a median age of 42 years (range 39 – 45 years) in comparison with the HIV-negative cohort with a median age of 54 years (range 53 – 55 years) . No difference was detected in disease phenotype when stage at presentation (p=0.7874), histological subtype (p=0.3375), grade of differentiation (p=0.8297), nodal involvement (p=0.0998) or hormone-receptor positivity (p=0.6285) was considered. Completion rates for systemic chemotherapy were excellent (>90%) regardless of HIV-status and no statistically significant toxicity was observed. The median CD4 count at diagnosis was 477 cells/μL (range 234 – 807 cells/μL), with a nadir value of 333 cells/μL (range 62 – 713 cells/μL), representing a decrease of 30.2% during treatment. One case of suspected treatment-related mortality was recorded. Conclusion: This retrospective study confirmed that women infected with HIV had a younger age at breast cancer diagnosis when compared to women with a negative HIV-status. No difference in disease phenotype could be demonstrated for women with HIV, denoting the coexistence of two common chronic diseases. Chemotherapy was tolerated well, but caused a median decline in CD4-count of 30% during treatment. / AFRIKAANSE OPSOMMING: Agtergrond: Infeksie met die Menslike Immuungebrek-Virus (MIV) is die pandemie van ons era, met ‘n geraamde 5.9 miljoen mense geïnfekteerd in Suid-Afrika volgens die Wêreld Gesondheids-Organisasie (WGO) in 2011. Kankers wat nie met MIV geassosieer word nie, het n belangrike oorweging in hierdie populasie-groep geword as gevolg van die gebruik van anti-retrovirale terapie wat die lewensverwagting van mense met MIV verleng. Navorsingsontwerp: ‘n Retrospektiewe kohort studie van alle vroue wat tydens die tydperk Januarie 2010 en Desember 2011 by die Gekombineerde Borskanker Kliniek te Tygerberg Hospitaal behandel was, verdeel in 3 groepe volgens hul retrovirale status. Metodes: ‘n Totaal van 819 vroue was oorweeg vir insluiting in die studie, waarvan 586 aan die insluitingskriteria voldoen het. Daar was 31 vroue met MIV (5.3%), 420 vroue het MIVnegatief getoets (71.7%), en 135 (23%) vroue waarvan die MIV-status onbekend was. Die fenotipe van borskanker was beskryf vir elke sub-groep, sowel as die toksiteite wat geassosieer word met die gebruik van standaard chemoterapie-skedules, insluitend die effek van chemoterapie op die CD4-telling. Bevindinge: Vroue met MIV het op ‘n statisties noemenswaardige (p<0.001) jonger ouderdom gepresenteer met borskanker (gemiddelde ouderdom 42 jaar, reikwydte 39 – 45 jaar) in vergelyking met vroue wat MIV-negatief was (gemiddelde ouderdom 54 jaar, reikwydte 53 – 55 jaar). Geen verskil was waargeneem in die fenotipe van borskanker in vroue met MIV vir die stadium by diagnose (p = 0.7874), histologiese tipe (p=0.3375), graad van differensiasie (p = 0.8297), nodale betrekking (p = 0.0998) of hormoon-reseptor status (p=0.6285) nie. Voltooing van sistemiese chemoterapie is bereik in meer as negentig persent van gevalle onafhanklik van MIV-status. ‘n Gemiddelde CD4-telling van 477 selle/μL (reikwydte 234 – 807 selle/ μL) met diagnose het ‘n gemiddelde afname van 30.2% tydens behandeling getoon na ‘n gemiddelde waarde van 333 selle/ μL (reikwydte 62 – 713 selle/ μL). Gevolgtrekkings: Hierdie retrospektiewe studie het bevind dat vroue met MIV op ‘n jonger ouderdom met borskanker presenteer as vroue wat MIV-negatief is. Geen noemenswaardige verskil was waargeneem in die fenotipe van borskanker in vroue met MIV nie, en dui daarop dat borskanker en MIV twee algemene, maar onafhanklike entiteite is. Chemoterapie was goed getolereer met ‘n gemiddelde afname in die CD4-telling van 30.2% tydens chemoterapie.