Mekanismen bag effekten af TGF-β3 på lavdosis-hypersensitivitet

Abstract

Celler med lav-dosis-hypersensitivitet bliver ikke arresteret i det såkaldt tidlige G2- kontrolpunkt, når de bestråles med doser mindre end en tærskelværdi, som er omkring 0.3 Gy for T-47D-celler. Det betyder at de celler, som er for tæt på mitosen til at kunne arresteres i det andet senere G2-kontrolpunkt (Sinclair-arrest), går i mitosen med ureparerede skader og dør. Hvis celler først primes med lav dosisrate (0.3 Gy/time) i en time, vil de inducere det tidlige G2-kontrolpunkt for doser helt ned til 0.1 Gy. Det betyder at lav-dosis- hypersensitiviteten forsvinder efter lav-dosisrate-priming. Faktisk ser man en overlevelsesrate som er højere for celler bestrålet med doser under 0.3 Gy end for ubestrålede celler (i det følgende kaldt oversving). Det er vist at TGF-β3 indgår i mekanismen bag effekten af LDR- primingen, og når man tilsætter rekombinant TGF-β3 til celler responderer de på samme måde som de lavdosisrate-primede celler. I dette projekt skulle effekten af TGF-β3 på lavdosishypersensitivitet i T-47D-celler undersøges. Dette blev delt i to problemstillinger. Den første var at undersøge om celler som fik tilsat TGF-β3 inducerede det tidlige G2-kontrolpunkt for doser under 0.3 Gy. Den anden var at undersøge om oversvinget kunne skyldes at celler i hvilefase (G0) stimuleres af TGF- β3 til at gå ind i cellecyklus. For at sikre præcis bestråling af cellerne blev der udført omfattende dosimetri på den røntgenapparatur, som blev brugt. Statistisk analyse af målingerne afdækkede flere usikkerheder i forbindelse med korte bestrålinger og en korrektionsfaktor på 2.0 ± 0.3% blev udledet i forbindelse med bestrålinger på 13 sekunder. Korrektionsleddet så ud til at være eksponentielt stigende jo kortere tid der bestråles. For 20 sekunders bestråling var korrektionsleddet på under 1%. Der blev udviklet en metode for at måle det mitotiske indeks i celler ved immunocytokemi med mitosemarkør anti-phospho-histone H3 og flowcytometri. Tilstrækkeligt mange af disse forsøg blev udført til at fastslå at TGF-β3 ikke påvirker det mitotiske indeks, når det tilsættes cellerne 16 timer før bestråling. Der blev også forsøgt udviklet en metode som brugte proliferationsmarkør KI-67 til at måle forholdet at senescente / quiescente celler. Dette studie fandt ingen beviser for at TGF-β3 aktiverer det tidlige G2-kontrolpunkt, når det tilsættes i rekombinant form til cellerne 16 timer før bestråling, selvom denne behandling fjerner lav-dosis-hypersensitiviteten.

Cells with low-dose hypersensitivity are not arrested at the so-called early G2 checkpoint when irradiated with doses lower than a 0.3 Gy in T-47D cells. This means that the cells that are too close to the mitosis to be arrested at the other G2 checkpoint (Sinclair arrest) enter mitosis with unrepaired damage and die. If the cells are primed at a low dose rate (0.3 Gy/hour for one hour), they will induce the early G2 checkpoint for doses as low as 0.1 Gy. This means that the low-dose hypersensitivity disappears following low dose rate (LDR) priming. In fact, one sees a survival rate which is higher for cells irradiated with doses below 0.3 Gy than that of unirradiated cells (hereinafter referred to as overshoot). TGF-β3 has been shown to be part of the mechanism behind the effect of LDR-priming, and when recombinant TGF-β3 is added to the cells, they respond in the same way as LDR-primed cells. In this project, the effect of TGF-β3 on low-dose hypersensitivity in T-47D cells was to be investigated. This was divided into two issues. The first was to investigate whether cells to which TGF-β3 was added induced the early G2 checkpoint for doses below 0.3 Gy. The second was to investigate whether the overshoot could be due to cells in the resting phase (G0) being stimulated by TGF-β3 to enter the cell cycle. To ensure accurate irradiation of the cells, extensive dosimetry was performed on the X-ray apparatus used. Statistical analysis of the measurements revealed several uncertainties associated with short exposures and a correction factor of 2.0 ± 0.3% was derived in the context of exposures of 13 seconds. The correction term appears to be exponentially increasing the shorter the exposure time. For 20 seconds of irradiation, the correction term was less than 1%. A method was developed to measure the mitotic index in cells by immunocytochemistry with mitotic marker anti-phospho-histone H3 and flow cytometry. Sufficiently many of these experiments were performed to determine that TGF-β3 does not affect the mitotic index when added to the cells 16 hours before irradiation. Another method was also attempted developed which used proliferation marker KI-67 to measure the ratio of senescent / quiescent cells. This study found no evidence that TGF-β3 activates the early G2 control point when added in recombinant form to the cells 16 hours before irradiation, although this treatment removes the low-dose hypersensitivity. 3