Examination of irradiated neuroblastoma and neuroepithelial cell lines for the interrelationship between cell survival, micronucleation, apoptosis and DNA repair

Akudugu, John Mbabuni (2000-12)

Thesis (Ph.D.)--Stellenbosch University, 2000.

Thesis

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.

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