Abstract
The Ph.D. thesis comprises two projects: (1) C/EBPα is dispensable for the ontogeny of PD1+ CD4+ memory T cells, but restricts their expansion in an age-dependent manner. (2) Modulation of RNR activity prolongs survival of mice with AML.
Immunosenescence is a condition of the immune system that refers to the gradual deterioration of the immune system caused by natural aging or development of disease. It is a condition that affects many different components of the immune system and results from changes in both the adaptive and innate immune system. One of the reasons for developing immunosenescence is believed to be changes in T cell distribution, in which the amount of naïve T cells decreases and the amount of memory T cells increases. Cancer cells express immunogenic antigens and mounting of the immune system can essentially recognize and defeat the cancer cells at the initial stages of the disease, however the cancer cells acquires several alternative mechanisms that protects them against this immuno surveillance. In C/EBPα-expressing PD-1+ MP CD4+ T cells, C/EBPα is suggested to be responsible for the senescent features of these cells, including impaired T cell receptor signaling and decreased proliferation. Fu rthermore, the amount of C/EBPα-expressing PD-1+ CD4+ T cells is increased during the development of leukemia, where they might account for the depressed T cell immuno-response that arises during development of disease.
The importance of C/EBPα expression for the development of age-dependent PD-1+ CD4+ T cells was therefore investigated as well as its importance for development of PD-1+ CD4+ T cells during leukemic development. My results showed that loss of C/EBPα expression in the lymphoid compartment led to an increased amount of aged PD-1+ CD4+ T cells, but not of young PD-1+ CD4+ T cells, suggesting that C/EBPα repress the accumulation of these cells in old mice. Furthermore, C/EBPα-deficiency in the lymphoid compartment had no effect on leukemic development and did not affect the accumulation of PD-1+ CD4+ T cells during development of leukemia. M data indicates that the anti-proliferative properties of C/EBPα are restricted to aged splenic PD-1+ CD4+ T cells and are otherwise not involved in regulating
proliferation or the transcriptional program of PD-1+ CD4+ T cells and does not affect development of myeloid leukemia.
The Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of
deoxyribonucleotides (dNTPs) to provide a strictly balanced supply of dNTPs required for DNA synthesis and DNA repair. The activity of this enzyme is under exquisite transcriptional and posttranscriptional control to avoid unbalanced dNTP pools that can lead to mis-incorporation of nucleotides into DNA, mutations, and cell death. The ribonucleotide reductase is a tetramer composed of two homodimeric subunits; R1:R2 when dNTPs are needed during DNA synthesis or R1:R2b when dNTPs are needed for DNA repair. The RNR genes are transcriptionally regulated during the cell cycle, in response to DNA damage, and its activity by allosteric regulation by nucleoside triphosphates and inhibitory proteins. In S. cerevisiae, Sml1 and Dif1 have been shown to bind to RNR and inhibit its activity, whereas in S. pombe the inhibitory protein is Spd1.
It has been demonstrated that the inability to degrade the RNR inhibitor Spd1 results in reduced dNTP pools and elevated mutation rates. Furthermore, S. cerevisiae and S. pombe yeast cells carrying a mutation that relaxes the allosteric feedback inhibition site of RNR display elevated dNTP pools and increased mutation rates. Taken together, this indicates that both reduced and elevated levels of dNTPs are mutagenic.
In order to investigate whether modulation of RNR activity affects tumor latency, MLLAF9 cells were transduced with retroviruses aimed at deregulating the ribonucleotide reductase, and then transplanted into lethally irradiated recipients. Since RNR subunits are highly conserved, we expected Sml1 and Dif1 to work across species barriers.
The results showed that overexpression of R2 and R2b as well as expression of Dif1 led to prolonged latency compared to control mice, suggesting that deregulation of RNR inhibits leukemic development. Expression of Sml1 in transplanted MLL-AF9 cells had no effect on the latency.
The distribution of myeloid lineage cells in the MLL-AF9-induced acute myeloid leukemias was not changed by overexpression of R2, or expression of Dif1 or Sml1. However, overexpression of R2b resulted in a more immature leukemia with more GMPs than control counterparts. When investigating the genetic profiles of sorted Gr-1+ cells from the leukemic mice, all Gr-1+ cells were highly similar among the different mice cohorts, indicating that these Gr-1+ cells are very alike and that overexpression of R2 and R2b and expression of Dif1 and Sml1 does not change the genetic profile of Gr-1+ cells.
Taken together, the results indicate that changes in RNR activity might inhibit tumor growth.
Immunosenescence is a condition of the immune system that refers to the gradual deterioration of the immune system caused by natural aging or development of disease. It is a condition that affects many different components of the immune system and results from changes in both the adaptive and innate immune system. One of the reasons for developing immunosenescence is believed to be changes in T cell distribution, in which the amount of naïve T cells decreases and the amount of memory T cells increases. Cancer cells express immunogenic antigens and mounting of the immune system can essentially recognize and defeat the cancer cells at the initial stages of the disease, however the cancer cells acquires several alternative mechanisms that protects them against this immuno surveillance. In C/EBPα-expressing PD-1+ MP CD4+ T cells, C/EBPα is suggested to be responsible for the senescent features of these cells, including impaired T cell receptor signaling and decreased proliferation. Fu rthermore, the amount of C/EBPα-expressing PD-1+ CD4+ T cells is increased during the development of leukemia, where they might account for the depressed T cell immuno-response that arises during development of disease.
The importance of C/EBPα expression for the development of age-dependent PD-1+ CD4+ T cells was therefore investigated as well as its importance for development of PD-1+ CD4+ T cells during leukemic development. My results showed that loss of C/EBPα expression in the lymphoid compartment led to an increased amount of aged PD-1+ CD4+ T cells, but not of young PD-1+ CD4+ T cells, suggesting that C/EBPα repress the accumulation of these cells in old mice. Furthermore, C/EBPα-deficiency in the lymphoid compartment had no effect on leukemic development and did not affect the accumulation of PD-1+ CD4+ T cells during development of leukemia. M data indicates that the anti-proliferative properties of C/EBPα are restricted to aged splenic PD-1+ CD4+ T cells and are otherwise not involved in regulating
proliferation or the transcriptional program of PD-1+ CD4+ T cells and does not affect development of myeloid leukemia.
The Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of
deoxyribonucleotides (dNTPs) to provide a strictly balanced supply of dNTPs required for DNA synthesis and DNA repair. The activity of this enzyme is under exquisite transcriptional and posttranscriptional control to avoid unbalanced dNTP pools that can lead to mis-incorporation of nucleotides into DNA, mutations, and cell death. The ribonucleotide reductase is a tetramer composed of two homodimeric subunits; R1:R2 when dNTPs are needed during DNA synthesis or R1:R2b when dNTPs are needed for DNA repair. The RNR genes are transcriptionally regulated during the cell cycle, in response to DNA damage, and its activity by allosteric regulation by nucleoside triphosphates and inhibitory proteins. In S. cerevisiae, Sml1 and Dif1 have been shown to bind to RNR and inhibit its activity, whereas in S. pombe the inhibitory protein is Spd1.
It has been demonstrated that the inability to degrade the RNR inhibitor Spd1 results in reduced dNTP pools and elevated mutation rates. Furthermore, S. cerevisiae and S. pombe yeast cells carrying a mutation that relaxes the allosteric feedback inhibition site of RNR display elevated dNTP pools and increased mutation rates. Taken together, this indicates that both reduced and elevated levels of dNTPs are mutagenic.
In order to investigate whether modulation of RNR activity affects tumor latency, MLLAF9 cells were transduced with retroviruses aimed at deregulating the ribonucleotide reductase, and then transplanted into lethally irradiated recipients. Since RNR subunits are highly conserved, we expected Sml1 and Dif1 to work across species barriers.
The results showed that overexpression of R2 and R2b as well as expression of Dif1 led to prolonged latency compared to control mice, suggesting that deregulation of RNR inhibits leukemic development. Expression of Sml1 in transplanted MLL-AF9 cells had no effect on the latency.
The distribution of myeloid lineage cells in the MLL-AF9-induced acute myeloid leukemias was not changed by overexpression of R2, or expression of Dif1 or Sml1. However, overexpression of R2b resulted in a more immature leukemia with more GMPs than control counterparts. When investigating the genetic profiles of sorted Gr-1+ cells from the leukemic mice, all Gr-1+ cells were highly similar among the different mice cohorts, indicating that these Gr-1+ cells are very alike and that overexpression of R2 and R2b and expression of Dif1 and Sml1 does not change the genetic profile of Gr-1+ cells.
Taken together, the results indicate that changes in RNR activity might inhibit tumor growth.
| Originalsprog | Engelsk |
|---|
| Forlag | Department of Biology, Faculty of Science, University of Copenhagen |
|---|---|
| Antal sider | 118 |
| Status | Udgivet - 2013 |