J. Peter McPherson

Brca1 required for T cell lineage development but not TCR loci rearrangement.

Brca1 (breast cancer1, early onset) deficiency results in early embryonic lethality. As Brca1 is highly expressed in the T cell lineage, a T cell–specific disruption of Brca1 was generated to assess the role of Brca1 in relation to T lymphocyte development. We found that thymocyte development in Brca1−/− mice was impaired not as a result of V(D)J T cell receptor (TCR) recombination but because thymocytes had increased expression of tumor protein p53. Chromosomal damage accumulation and abnormal cell death were observed in mutant cells. We found that cell death inhibitor Bcl-2 overexpression, or p53−/− backgrounds, completely restored survival and development of Brca1−/− thymocytes; peripheral T cell numbers were not totally restored in Brca1−/− p53−/− mice; and that a mutant background for p21 (cyclin-dependent kinase inhibitor 1A) did not restore Brca1−/− thymocyte development, but partially restored peripheral T cell development. Thus, the outcome of Brca1 deficiency was dependent on cellular context, with the major defects being increased apoptosis in thymocytes, and defective proliferation in peripheral T cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin poly(ADP-ribose) polymerase (TiPARP, ARTD14) is a mono-ADP-ribosyltransferase and repressor of aryl hydrocarbon receptor transactivation

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly(ADP-ribose) polymerase (TiPARP/ARTD14) is a member of the PARP family and is regulated by the aryl hydrocarbon receptor (AHR); however, little is known about TiPARP function. In this study, we examined the catalytic function of TiPARP and determined its role in AHR transactivation. We observed that TiPARP exhibited auto-mono-ADP-ribosyltransferase activity and ribosylated core histones. RNAi-mediated knockdown of TiPARP in T-47D breast cancer and HuH-7 hepatoma cells increased TCDD-dependent cytochrome P450 1A1 (CYP1A1) and CYP1B1 messenger RNA (mRNA) expression levels and recruitment of AHR to both genes. Overexpression of TiPARP reduced AHR-dependent increases in CYP1A1-reporter gene activity, which was restored by overexpression of AHR, but not aryl hydrocarbon receptor nuclear translocator. Deletion and mutagenesis studies showed that TiPARP-mediated inhibition of AHR required the zinc-finger and catalytic domains. TiPARP and AHR co-localized in the nucleus, directly interacted and both were recruited to CYP1A1 in response to TCDD. Overexpression of Tiparp enhanced, whereas RNAi-mediated knockdown of TiPARP reduced TCDD-dependent AHR proteolytic degradation. TCDD-dependent induction of AHR target genes was enhanced in Tiparp−/− mouse embryonic fibroblasts compared with wildtype controls. Our findings show that TiPARP is a mono-ADP-ribosyltransferase and a transcriptional repressor of AHR, revealing a novel negative feedback loop in AHR signalling.

In Search of a Function for BCLAF1

BCLAF1 was originally identified as a protein that interacts with antiapoptotic members of the Bcl2 family. Initial studies indicated a role for this protein as an inducer of apoptosis and repressor of transcription. Subsequent studies have shown that BCLAF1 plays criticals roles in a wide range of processes that are not normally associated with actions of Bcl2 family members, including lung development, T-cell activation, and control of the lytic infection program of Kaposis sarcoma–associated herpesvirus. Here, we provide an overview of findings from past studies that both support and challenge the role of BCLAF1 in cell death and transcriptional control. We also present recent findings from our laboratory and others indicating a role for BCLAF1 in post-transcriptional processes that impact mRNA metabolism, instead of a direct role for this protein in apoptosis or transcription.

Essential role for Bclaf1 in lung development and immune system function

Bcl-2 associated factor 1 (Bclaf1) is a nuclear protein that was originally identified in a screen of proteins that interact with the adenoviral bcl-2 homolog E1B19K. Overexpression of Bclaf1 was shown to result in apoptosis and transcriptional repression that was reversible in the presence of Bcl-2 or Bcl-xL. Furthermore, antiapoptotic members, but not proapoptotic members of the Bcl-2 protein family, were shown to interact with Bclaf1 and prevent its localization to the nucleus. Bclaf1 has also recently been identified as a binding partner for Emerin, a nuclear membrane protein that is mutated in X-linked recessive Emery–Dreifuss muscular dystrophy. To ascertain the in vivo function of Bclaf1, we have generated mice that carry a targeted mutation of the bclaf1 locus. In this study, we show that Bclaf1 is required for proper spatial and temporal organization of smooth muscle lineage during the saccular stage of lung development. We also show that Bclaf1 is dispensable for thymocyte development but is essential for peripheral T-cell homeostasis. Despite its postulated role as a proapoptotic protein, Bclaf1-deficient cells did not show any defect in cell death linked to development or after exposure to various apoptotic stimuli. Our findings show a critical role for Bclaf1 in developmental processes independent of apoptosis.

Role of proteasomal degradation in the cell cycle-dependent regulation of DNA topoisomerase IIα expression☆ ☆

1DNA topoisomerase II (topo II) is a nuclear enzyme that modifies DNA topology and also serves as a target to mediate the cytotoxicity of several antineoplastic agents. Several reports have demonstrated that a reduction of topo II is associated with reduced sensitivity to these agents. Topo II exists as two isoforms in mammalian cells: topo IIalpha and topo IIbeta. In MCF-7 cells, the half-life (mean +/- SEM) values of topo IIalpha and topo IIbeta in situ were 6.6 +/- 0.3 and 17.6 +/- 2.3 hr, respectively, as determined by [(35)S]methionine/cysteine pulse-chase analysis. Degradation of topo IIalpha in situ was abrogated by the presence of proteasome inhibitors, and the relative activities were carbobenzoxy-leucyl-leucyl-leucinal (MG132) > carbobenzoxy-leucyl-leucyl-norvalinal (MG115) > ALLN congruent with lactacystin. ATP-dependent degradation of topo IIalpha, but not topo IIbeta, was observed in extracts of asynchronously dividing HeLa and MCF-7 cells. Furthermore, degradation of topo IIalpha was abrogated by the proteasome inhibitors MG132 and MG115, but not by lactacystin, in extracts of asynchronously dividing MCF-7 cells. Finally, degradation of topo IIalpha, but not topo IIbeta, was observed to occur in a cell cycle-dependent fashion, in extracts of synchronized HeLa cells, with maximal loss of the alpha isoform occurring 2 hr after release from mitotic arrest. This degradation of topo IIalpha appeared to be facilitated by an ATP-dependent activity. Furthermore, high molecular weight bands (>200 kDa), which may represent polyubiquitinated-topo IIalpha conjugates, were also detected in extracts of synchronized HeLa cells. This study provides evidence for a role of the ubiquitin-proteasome pathway in the cell cycle-dependent regulation of topo IIalpha expression.

p53 gene status and chemosensitivity of childhood acute lymphoblastic leukemia cells to adriamycin

The role of p53 as a determinant of sensitivity of ten childhood acute lymphoblastic leukemia (ALL) cell lines to Adriamycin (ADR) was investigated. ADR-sensitive cell lines were found to have wild-type (wt) p53, whereas resistant cell lines contained point mutations in the gene. The basal level of wt p53 protein in sensitive cells was lower than that of mutant p53 in resistant cells, however, after ADR treatment a 6- to 20-fold dose-dependent increase in wt p53 was observed, whereas mutant p53 increased only twofold. The percentage of apoptotic cells in ADR-sensitive lines with wt p53 ranged from 43 to 93% following ADR treatment, whereas that in resistant lines with mutant p53 was only 8-13%. The ratio of constitutive levels of Bax/Bcl-2 was significantly higher in cells containing wt p53 than in cells with mutant p53. These results suggest that p53 gene status and the ability of p53 to induce apoptosis may be determinants of sensitivity to ADR in childhood ALL cells.

A role for Mus81 in the repair of chromium-induced DNA damage

Hexavalent chromium (Cr[VI]) is a toxic environmental contaminant that is capable of producing a broad spectrum of DNA damage. The ability of Cr[VI] to induce mutagenesis and neoplastic transformation has been attributed to its genotoxic action, however our understanding of molecular mechanisms involved in the repair of Cr[VI]-induced DNA damage remains incomplete. Here, we report that Mus81, an enzyme that participates with Eme1 in the resolution of replication fork damage caused by certain lesions, is involved in the repair of Cr[VI]-induced DNA damage. Mus81-deficient cells were found to be more susceptible to Cr[VI]-induced proliferation arrest and more sensitive to the long-term cytotoxic effects of Cr[VI] than isogenic wild-type cells. Following Cr[VI] exposure, Mus81-deficient cells displayed a lag in the disappearance of Rad51 foci, exhibited elevated replication-associated gamma-H2AX and showed an increased incidence of chromosomal instability compared to wild-type cells. Our findings support a role for Mus81 in the resolution of replication-associated DNA damage associated with this genotoxic agent, by converting Cr[VI]-DNA lesions into a form more amenable for homologous recombination.

N-Hydroxylation of 4-Aminobiphenyl by CYP2E1 Produces Oxidative Stress in a Mouse Model of Chemically Induced Liver Cancer

4-Aminobiphenyl (ABP) is a trace component of cigarette smoke and hair dyes, a suspected human carcinogen and a potent rodent liver carcinogen. Postnatal exposure of mice to ABP results in a higher incidence of liver tumors in males than in females, paralleling the sex difference in human liver cancer incidence. A traditional model of ABP tumorigenesis involves initial CYP1A2-mediated N-hydroxylation, which eventually leads to production of mutagenic ABP-DNA adducts that initiate tumor growth. However, several studies have found no correlation between sex or CYP1A2 function and the DNA-damaging, mutagenic, or tumorigenic effects of ABP. Oxidative stress may be an important etiological factor for liver cancer, and it has also been linked to ABP exposure. The goals of this study were to identify novel enzyme(s) that contribute to ABP N-oxidation, and to investigate a potential role for oxidative stress in ABP liver tumorigenicity. Isozyme-selective inhibition experiments using liver microsomes from wild-type and genetically modified mice identified CYP2E1 as a major ABP N-hydroxylating enzyme. The N-hydroxylation of ABP by transiently expressed CYP2E1 produced oxidative stress in cultured mouse hepatoma cells. In vivo postnatal exposure of mice to a tumorigenic dose of ABP also produced oxidative stress in male wild-type mice, but not in male Cyp2e1(-/-) mice or in female mice. However, a stronger NRF2-associated antioxidant response was observed in females. Our results identify CYP2E1 as a novel ABP-N-oxidizing enzyme, and suggest that sex differences in CYP2E1-dependent oxidative stress and antioxidant responses to ABP may contribute to the observed sex difference in tumor incidence.

Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair

Individuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair. The relationship of this repair defect to disease traits remains unclear, given that crosslink sensitivity is recapitulated in FA mouse models without most of the other disease-related features. Mice deficient in Mus81 are also defective in crosslink repair, yet MUS81 mutations have not been linked to FA. Using mice deficient in both Mus81 and the FA pathway protein FancC, we show both proteins cooperate in parallel pathways, as concomitant loss of FancC and Mus81 triggered cell-type-specific proliferation arrest, apoptosis and DNA damage accumulation in utero. Mice deficient in both FancC and Mus81 that survived to birth exhibited growth defects and an increased incidence of congenital abnormalities. This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity. Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.