Bo LinFollow

Date of Completion

5-25-2017

Embargo Period

12-31-2017

Major Advisor

Christopher D. Heinen

Associate Advisor

Sandra K. Weller

Associate Advisor

Ann Cowan

Associate Advisor

Stormy J. Chamberlain

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

The DNA mismatch repair (MMR) pathway is a very important DNA repair pathway to maintain genomic integrity. Germline mutations in the MMR genes can cause a hereditary cancer predisposition syndrome, Lynch Syndrome (LS). LS patients develop colorectal cancer as well as other extracolonic cancers at an early age. However, how the loss of DNA MMR leads to tumorigenesis remains unclear. The MMR mediated DNA damage response to the alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) observed in various cancer cell lines may contribute to preventing tumorigenesis by eliminating damaged cells. In the first part of this study, we examined the MMR dependent DNA damage response in the human pluripotent stem cell (hPSC) which is a nontransformed cell model. We found that hPSCs are hypersensitive to alkylation damage which triggers massive apoptosis. Interestingly, the nature of this alkylation response differs from that previously reported in somatic cells. In somatic cells, a permanent G2/M cell cycle arrest is induced in the second cell cycle after DNA damage. The hPSCs, however, directly undergo apoptosis in the first cell cycle. Furthermore, the signaling mechanisms of this damage response are also very different from somatic cells in that the checkpoint kinases Chk1 and Chk2 are not activated in hPSCs in response to alkylation damage, but rather p53 activation is responsible for inducing apoptosis. This response reveals that hPSCs rely on apoptotic cell death as an important defense to avoid mutation accumulation.

Since LS patients predominantly develop colorectal cancer and human embryonic stem cells (hESCs) can be differentiated into intestinal organoids in vitro, in the second part of this study we generated both hESCs-derived human intestinal organoids (HIOs) and adult human intestinal enteroids (HIEs) from patient colon samples to study the damage responses to alkylation damage in intestinal cells specifically. We found that the MMR pathway can direct multiple responses to DNA damage in different intestinal cell types in HIOs. Intestinal stem cells (ISCs) appear more prone to undergo apoptosis in response to DNA damage whereas more differentiated cells such as the transient amplifying cells are more likely to senesce. Both mechanisms may play an important role in tumor suppression by eliminating or halting progression of damaged cells. Therefore loss of MMR pathway function might provide an immediate selective advantage at an early stage during tumorigenesis in LS patients. Taken together, this work further reveals the MMR-dependent DNA damage response in nontransformed cell types and cell types related to LS, and provides insights into how loss of these damage responses may contribute to tumorigenesis at an early stage in LS patients.

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