Role of the Cell Cycle Regulator Cdh1 in Physiology and Pathology of Hematopoiesis

Abstract 2419

Various key molecules for cell cycle, especially G0/G1 regulators, have effects not only on cell proliferation but also on cell differentiation. Cdh1, one of the co-activators for anaphase-promoting complex/cyclosome, plays a crucial role in the mitotic phase, but has recently been identified as a G0/G1 regulator, suggesting that the role of Cdh1 in cell differentiation. Because there are only few reports about Cdh1 from this point of view, we focused on Cdh1 functions on the hematopoietic system, in which distinct populations of cells can be precisely identified by their cell surface markers, in physiology and pathology.

For this purpose, we generated Cdh1 conditional gene-trap (GT) mice, by overcoming the embryonic lethality of Cdh1 homozygous GT mice. We introduced the Cdh1 cDNA replacing vector into ES cells derived from Cdh1 heterozygous GT mice. The resulted construct contains the floxed Cdh1 cDNA allele which is cleaved under the existence of Cre recombinases. We crossed mice carrying this Cdh1 transgene in homozygous (Cdh1^f/f) with Mx1-Cre transgenic mice to obtain Mx1-Cre (+) / Cdh1^f/f mice, in which Cre recombinases are induced in vivo by administration of pIpC.

In this system, we found that the Cdh1-deficient mice 4 months after pIpC treatment, compared to Cdh1-intact mice (Mx1-Cre (-) / Cdh1^f/f mice), exhibited a subtle but significant decrease in absolute number of mature lineage progenitor cells (4.3 ± 0.31 × 10⁷ vs 3.2 ± 0.10 × 10⁷ /femurs and tibiae; p=0.009). Furthermore, this phenomenon was conspicuous by irradiation as short as 7 days after pIpC treatment. In 48 hours post-irradiation, the absolute number of mature lineage progenitor cells decreased markedly in the Cdh1-deficient mice (7.4 ± 0.82 × 10⁶ vs 3.6 ± 0.46 × 10⁶; p=0.0023) and in addition, both of CD34+ and CD34- LSK cells were also decreased (absolute number of CD34- cells: 905 ± 194 vs 344 ± 223; p= 0.03). These results indicate that the loss of Cdh1 induces genotoxic fragility especially in these two subpopulations, the mature lineage progenitors and the stem cells. We also confirmed that the increased cell loss induced by irradiation in Cdh1-deficient mice is the result of mitotic catastrophe following G2/M checkpoint slippage due to loss of Cdh1 by DNA content analysis.

We next focused on how oncogenic stress, as another genotoxic stress, effects on the cell fragility by Cdh1 loss. We performed retroviral transduction of N-myc into Cdh1-intact and Cdh1-deficient bone marrow mononuclear cells (BM-MNCs) and transplanted those into irradiated wild type mice. In this system, which our laboratory has established recently, the transplanted mice develop precursor B cell lymphoblastic leukemia (pre-B ALL) phenotype in high frequency (more than 80%) when wild type BM-MNCs were used as cell source. Our hypothesis at that time was that oncogenic stress due to N-myc induces the loss of stem/progenitor cell function, and in result, that Cdh1 loss reveals negative effects on leukemogenesis or changes its lineage phenotype by affecting pseudodifferentiation due to N-myc. However, against our speculation, 70% (7 out of 10) of mice transplanted with N-myc transduced Cdh1-deficient BM-MNCs developed pre-B ALL, which was the same frequency and the same phenotype as in Cdh1-intact cell sources. Of note, Cdh1 loss did not have a great impact on the prognosis of these pre-B ALL mice (median survival: 80 days in Cdh1-intact group vs 95 days in Cdh1-deficient group; p= 0.049).

In conclusion, our results suggest that Cdh1 regulates the pool sizes of the hematopoietic stem cells and mature lineage progenitor cells both physiologically and pathologically; especially under irradiation stress. In contrast, Cdh1 is dispensable for B cell leukemogenesis and does not have a great impact on the natural prognosis of non-treated pre-B ALL. It is interesting that oncomine mRNA microarray database and other few reports indicate that human pre-B ALL cases are also divided into two groups according to the expression level of Cdh1, and it is the matter remained to be solved whether Cdh1 expression level affects the prognosis of treated patients. We propose that our Cdh1-deficient pre-B ALL mice have a potential as promising mouse model in order to assess this proposition and to prove that Cdh1 affects the sensitivity of pre-B ALL to treatments which causes the genotoxic stress, such as radiotherapy and genotoxic agents.