Pretorius, G. H. J.Van den Heever, Wilhelmina Maria Jacoba2018-03-232018-03-232001-11http://hdl.handle.net/11660/8066English: Squamous cell carcinoma of the esophagus is a cancer with a high incidence in South Africa. We have investigated the prognostic value of telomerase activity in tumors as well as in nearby normal tissue. Biopsies from 98 patients were analyzed using an adaptation of the TRAP assay. We found all tumor biopsies to have moderate to high telomerase activity, while one third of biopsies from normal mucosa were negative. The telomerase activity level of the tumors had no prognostic value (P=0.95) as determined by the log rank test. A P-value of 0.02 was found when the telomerase-negative and moderately positive normal biopsies were grouped together and compared to those with high activity. Our results show that telomerase activity of normal mucosa in the vicinity of the tumor can identify a population of patients with significantly worse prognosis, even in late stage patients. Telomerase has attracted intense interest as a possible target for cancer therapeutics. Previous attempts at inhibiting telomerase activity utilized antisense oligonucleotides targeted at the template region of the RNA subunit of the enzyme. Although it worked well in cell extracts, getting the oligonucleotides into intact cells are difficult. We attempted a peptide-based approach to overcome the transport problem. A phage-display selection strategy was designed to isolate peptides (7 and 12 amino acids) capable of binding to the RNA template with high affinity and in so doing preventing the enzyme from elongating existing telomeres. Both libraries showed no high affinity binding to the RNA. The most probable explanation is that such short peptides are incapable of binding sequence-specifically to nucleic acids. Many studies have indicated the importance of telomerase activity as an independent prognostic indicator in a variety of cancers, but recent results have shown that telomere length is actually a better indicator, as it shows the final result oftelomerase activity. We thus decided to develop a flow cytrometric method that would allow us to analyze telomere length in small tissue samples. After the initial technique had been established on lymphocytes, it was tested on the SNO cell line before we switched to solid tissue. The background of cellular debris and cell clusters was much worse than for lymphocytes, but differences in signal strength could be seen. From the results obtained it is clear that this method is not optimized yet. The results also indicate that at this stage of development, flow-FISH is inferior to telomerase activity as prognostic indicator. The best way to validate the current method is to analyze the same samples by Southern hybridization and flow-FISH, preferably well defined tumor and normal tissue.Afrikaans: Plaveiselepiteelkarsinoom is een van die volopste kankers in Suid-Afrika, veral in swart mans. Hierdie projek is deel van 'n nasionale navorsingsprogram om die oorsake, verloop en behandeling van die siekte te bestudeer. Daar is op telomere en telomerase gekonsentreer aangesien daar geen inligting hieroor bekend was toe die studie begin het me. Die moontlik prognostiese waarde van telomerase aktiwiteit in tumorbiopsies en in naasliggende normale weefsel, is ondersoek. Die telomerase aktiwiteitsvlakke van tumorweefsel het geen korrelasie met oorlewing gehad nie (75 pasiënte, P=0.95). Indien die normale weefsel verdeel word in hoë aktiwiteit vs matig/negatief, het 19 groep 'n betekenisvolle langer oorlewing as die met hoë aktiwiteit (P=0.02). Die telomerase aktiwiteit van die normale weefsel mag 'n vroeë aanduiding wees van verspreiding. Telomerase word ook beskou as 'n moontlike terapeutiese teiken, aangesien inhibisie van die ensiem in tumorsellyne lei tot seldood. Die meeste inhibitore is oligonukleotiede of PNAs gemik op die templaatgebied van die ensiem se RNA-subeenheid. Hoewel dit goed gewerk het in vitro, is transport van die molekules na die kern 'n probleem. Ons het dus inhibitore gesoek in 2 peptiedbiblioteke (7 en 12 aminosure) wat op die oppervlak van bakteriofage geanker is. Die teiken was die telomerase RNA templaat wat mbv in vitro transkipsie vervaardig is. Ongelukkig was daar geen peptiede wat die RNA met hoë affiniteit gebind het nie, waarskynlik omdat die peptiedgroottes kleiner is as die minimum wat vir RNA binding benodig word. Telomeerlengte is die produk van telomerase aktiwiteit, daarom het ons besluit om te kyk of daar veranderings in lengte is tussen tumor en normale weefsel. Aangesien die biopsies uiters klein is, kon ons nie Southernhibridisasie daarop doen nie en moes 'n alternatiewe metode ontwikkel word. Vloeisitometrie is gekies, aangesien dit 'n kragtige tegniek is waarmee individuele selle bestudeer kan word. Die homogenisering van soliede weefsel was egter 'n probleem wat eers oorkom moes word. Die metode soos dit nou daar uitsien maak gebruik van 'n kollagenaseverteringstap om die weefsel af te breek, dit geskied in 'n lisebuffer wat die plasmamembrane laat breek, maar die kernmemembrane behou. Die kernsuspensie word dan verhit om die DNA te denatureer en gehibridiseer met 'n FITC-gemerkte PNA peiler wat uit telomeerherhalings bestaan. Die DNA in die kern word gekleur met 7 AAD en die suspensie word deur 'n vloeisitometer gestuur. Die metode is stapsgewys op limfosiete, weefselkultuurselle, bobbejaanlewer en uiteindelik esofagusbiopsies ontwikkel. Daarna is gepaarde biopsies (normaal en tumor) van 70 pasiënte geanaliseer. Die resultate van hierdie eksperimente moet as voorlopig beskou word, aangesien die metode nog nie gevalideer is deur vergelyking met Southernhibridisasie nie. Desnieteenstaande is daar aanduidings dat die gemiddelde telomeerlengte afneem tussen normale en tumorweefsel. Verdere afleidings sal moet wag totdat die metode finaal gevalideer is.enEsophageal cancerEpitheliumGastroscopyTelomeresTelomerasePhage display technologyFlow-fishEsophagus -- CancerThesis (Ph.D.(Haematology and Cell Biology))--University of the Free State, 2001DNA polymerasesSquamous cell carcinomaThesisUniversity of the Free State