New Oncolytic Adenoviruses with Hypoxia- and Estrogen Receptor-Regulated Replication

Oncolytic adenoviruses with restricted replication can be produced if the expression of crucial transcription units of the virus is controlled by tissue- or tumor-specific promoters. Here we describe a method for the rapid incorporation of exogenous promoters into the E1A and E4 regions of the human adenovirus type 5 genome. Using this system, we have generated AdEHT2 and AdEHE2F, two conditionally replicative adenoviruses for the treatment of breast cancer. The expression of the E1A gene in both viruses is controlled by a minimal dual-specificity promoter that responds to estrogens and hypoxia. The tight regulation of E1A expression correlated with the ability of these viruses to replicate and kill human cancer cells that express estrogen receptors, or are maintained under hypoxic conditions. The telomerase reverse transcriptase (TERT) promoter and the E2F-1 promoter are preferentially activated in cancer cells. They were introduced into the E4 region of AdEHT2 and AdEHE2F, respectively. The telomerase core promoter failed to block the replication of the virus in telomerase-negative cells. In contrast, AdEHE2F was attenuated in nontransformed quiescent cells growing under normoxic conditions, suggesting that an intact pRB pathway with low levels of E2F transcription factors acts as a negative modulator for the virus. These data indicate that the simultaneous regulation of E1A and E4 viral transcription units by the appropriate combination of promoters can increase the tumor selectivity of oncolytic adenoviruses.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63195/1/104303402760293574.pd

Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells

A central issue in stem cell biology is to understand the mechanisms that regulate the self-renewal of haematopoietic stem cells (HSCs), which are required for haematopoiesis to persist for the lifetime of the animal(1). We found that adult and fetal mouse and adult human HSCs express the proto-oncogene Bmi-1. The number of HSCs in the fetal liver of Bmi-1(-/-) mice(2) was normal. In postnatal Bmi-1(-/-) mice, the number of HSCs was markedly reduced. Transplanted fetal liver and bone marrow cells obtained from Bmi-1(-/-) mice were able to contribute only transiently to haematopoiesis. There was no detectable self-renewal of adult HSCs, indicating a cell autonomous defect in Bmi-1(-/-) mice. A gene expression analysis revealed that the expression of stem cell associated genes(3), cell survival genes, transcription factors, and genes modulating proliferation including p16(Ink4a) and p19(Arf) was altered in bone marrow cells of the Bmi-1(-/-) mice. Expression of p16(Ink4a) and p19(Arf) in normal HSCs resulted in proliferative arrest and p53-dependent cell death, respectively. Our results indicate that Bmi-1 is essential for the generation of self-renewing adult HSCs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62508/1/nature01587.pd

A Novel, Conditionally Replicative Adenovirus for the Treatment of Breast Cancer That Allows Controlled Replication of E1a-Deleted Adenoviral Vectors

The efficiency of gene therapy strategies against cancer is limited by the poor distribution of the vectors in the malignant tissues. To solve this problem, a new generation of tumor-specific, conditionally replicative adenoviruses is being developed. To direct the replication of the virus to breast cancer, we have considered one characteristic present in a great proportion of these cancers, which is the expression of estrogen receptors (ERs). On the basis of the wild-type adenovirus type 5, we have constructed a conditionally replicative adenovirus (Ad5ERE2) in which the E1a and E4 promoters have been replaced by a portion of the pS2 promoter containing two estrogen-responsive elements (EREs). This promoter induces transcriptional activation of the E1a and E4 units in response to estrogens in cells that express the ERs. Ad5ERE2 is able to kill ER+ human breast cancer cell lines as efficiently as the wild-type virus, but has decreased capacity to affect ER- cells. By complementation of the E1a protein in trans, Ad5ERE2 allows restricted replication of a conventional E1adeleted adenoviral vector. When a virus expressing the proapoptotic gene Bc1-xs (Clarke et al., Proc. Natl. Acad. Sci. U.S.A. 1995;92:11024-11028) is used in combination with Ad5ERE2, the ability of both viruses to induce cell death is dramatically increased, and the effect can be modulated by addition of the antiestrogen tamoxifen.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63245/1/10430340050143435.pd

Long-term haematopoietic reconstitution by Trp53(-/-)p16(Ink4a-/-)p19(Arf-/-) multipotent progenitors

Haematopoiesis is maintained by a hierarchical system where haematopoietic stem cells ( HSCs) give rise to multipotent progenitors, which in turn differentiate into all types of mature blood cells1. HSCs maintain themselves for the lifetime of the organism because of their ability to self- renew. However, multipotent progenitors lack the ability to self- renew, therefore their mitotic capacity and expansion potential are limited and they are destined to eventually stop proliferating after a finite number of cell divisions(1,2). The molecular mechanisms that limit the proliferation capacity of multipotent progenitors and other more mature progenitors are not fully understood(2,3). Here we show that bone marrow cells from mice deficient in three genes genetically downstream of Bmi1-p16(Ink4a), p19(Arf) and Trp53 ( triple mutant mice; p16(Ink4a) and p19(Arf) are alternative reading frames of the same gene ( also called Cdkn2a) that encode different proteins) - have an approximately 10-fold increase in cells able to reconstitute the blood long term. This increase is associated with the acquisition of long- term reconstitution capacity by cells of the phenotype c-kit(+)Sca-1(+)Flt3(+)CD150(-)CD48(-)Lin(-), which defines multipotent progenitors in wild- type mice(4-6). The pattern of triple mutant multipotent progenitor response to growth factors resembles that of wild- type multipotent progenitors but not wild- type HSCs. These results demonstrate that p16(Ink4a)/p19(Arf) and Trp53 have a central role in limiting the expansion potential of multipotent progenitors. These pathways are commonly repressed in cancer, suggesting a mechanism by which early progenitor cells could gain the ability to self- renew and become malignant with further oncogenic mutations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62767/1/nature06869.pd