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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 cells (1). HSCs [...]

Author(s): In-kyung Park [1]; Dalong Qian [1]; Mark Kiel [2]; Michael W. Becker [1]; Michael Pihalja [1]; Irving L. Weissman [3]; Sean J. Morrison [2]; Michael F. Clarke (corresponding author) [...]

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 cells. 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. The molecular mechanisms that limit the proliferation capacity of multipotent progenitors and other more mature progenitors are not fully understood. Here we show that bone marrow cells from mice deficient in three genes genetically downstream of Bmi1—p16Ink4a, p19Arf and Trp53 (triple mutant mice; p16Ink4a and p19Arf 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. 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 p16Ink4a/p19Arf 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. [ABSTRACT FROM AUTHOR]

Objectives: Antigen-specific CD4(+) T cells epigenetically modified with DNA methylation inhibitors overexpress genes normally suppressed by this mechanism, including CD11a, CD70, CD40L and the KIR gene family. The altered cells become autoreactive, losing restriction for nominal antigen and responding to self-class II major histocompatibility complex (MHC) molecules without added antigen, and are sufficient to cause a lupus-like disease in syngeneic mice. T cells overexpressing the same genes are found in patients with active lupus. Whether these genes are co-overexpressed on the same or different cells is unknown. The goal of this study was to determine whether these genes are overexpressed on the same or different T cells and whether this subset of CD4(+) T cells is also present in patients with lupus and other rheumatic diseases., Methods: Multicolour flow cytometry was used to compare CD11a, CD70, CD40L and KIR expression on CD3(+)CD4(+)CD28(+) T cells to their expression on experimentally demethylated CD3(+)CD4(+)CD28(+) T cells and CD3(+)CD4(+)CD28(+) T cells from patients with active lupus and other autoimmune diseases., Results: Experimentally demethylated CD4(+) T cells and T cells from patients with active lupus have a CD3(+)CD4(+)CD28(+)CD11a(hi)CD70(+)CD40L(hi)KIR(+) subset, and the subset size is proportional to lupus flare severity. A similar subset is found in patients with other rheumatic diseases including rheumatoid arthritis, systemic sclerosis and Sjögren's syndrome but not retroperitoneal fibrosis., Conclusions: Patients with active autoimmune rheumatic diseases have a previously undescribed CD3(+)CD4(+)CD28(+)CD11a(hi)CD70(+)CD40L(hi)KIR(+) T cell subset. This subset may play an important role in flares of lupus and related autoimmune rheumatic diseases, provide a biomarker for disease activity and serve as a novel therapeutic target for the treatment of lupus flares.

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.