In vivo potential effects of adenovirus type 5 E1A and E1B on lung carcinogenesis and lymphoproliferative inflammation.

Triggering uncontrolled cellular proliferation, chronic inflammation, and/or disruption of p53 activity is critical for tumorigenesis initiated by latent viral oncogenes. The adenovirus type 5 (Ad5) early genes E1A and E1B can maintain lifelong latency in the lungs of patients with chronic pulmonary diseases. To determine the in vivo effects of the latent Ad5 E1A and E1B oncogenes, we have examined the influence of Ad5 E1A and E1B gene products on mouse lung carcinogenesis and inflammation by generation and characterization of lung-specific transgenic mouse models. Here, we show that either the Ad5 E1A 243-amino-acid (aa) protein or the E1B 58-kDa protein was dominantly expressed in the transgenic lung. Preferential expression of Ad5 E1A 243-aa protein alone was not sufficient to induce lung carcinogenesis but resulted in low-grade cellular proliferation and high-grade lymphoproliferative inflammation in the lung. The presence of Ad5 E1B dramatically enhanced the expression of the E1A 243-aa protein, in addition to impairing p53 and apoptosis response, resulting in uncontrolled cellular proliferation, lymphoproliferative inflammation, and metastatic carcinomas in the lung after a period of latency. Our studies may provide clues to understanding the potential in vivo biological effects of Ad5 E1A and E1B latent in the lung and a new scope for assessing in vivo functions of viral genes latent in the infection target tissue.

Disease model: Fanconi anemia.

Fanconi anemia (FA) is a chromosomal instability syndrome characterized by the presence of pancytopenia, congenital malformations and cancer predisposition. Six genes associated with this disorder have been cloned, and mice with targeted disruptions of several of the FA genes have been generated. These mouse models display the characteristic FA feature of cellular hypersensitivity to DNA cross-linking agents. Although they do not develop hematological or developmental abnormalities spontaneously, they mimic FA patients in their reduced fertility. Studies using these animal models provide valuable insights into the involvement of apoptotic pathways in FA, and help characterize the defects in FA hematopoietic cells. In addition, mouse models are also useful for testing treatments for FA.

Transgenic expression of CECR1 adenosine deaminase in mice results in abnormal development of heart and kidney.

Cat eye syndrome is a rare developmental defect associated with duplication of chromosome 22q11. The patients demonstrate specific abnormalities of heart, kidney, and eye. Here we attempted to produce a model for this defect by expressing CECR1 adenosine deaminase, a gene duplicated in cat eye syndrome patients, in mice. The transgenic mice expressed CECR1 under the control of either beta-actin promoter for ubiquitous expression or myosin heavy chain for heart-specific expression. The transgenics expressing CECR1 in the heart demonstrated high rate of embryonic and neonatal lethality. The mice from all the lines examined showed enlargement of the heart. Abnormalities of the kidney and eye were also detected in mice expressing CECR1 under control of the actin promoter.

FANCC, FANCE, and FANCD2 form a ternary complex essential to the integrity of the Fanconi anemia DNA damage response pathway.

Fanconi anemia (FA) is a genetically heterogeneous disorder characterized by bone marrow failure, cancer predisposition, and increased cellular sensitivity to DNA-cross-linking agents. The products of seven of the nine identified FA genes participate in a protein complex required for monoubiquitination of the FANCD2 protein. Direct interaction of the FANCE protein with both fellow FA complex component FANCC and the downstream FANCD2 protein has been observed in the yeast two-hybrid system. Here, we demonstrate the ability of FANCE to mediate the interaction between FANCC and FANCD2 in the yeast three-hybrid system and confirm the FANCE-mediated association of FANCC with FANCD2 in human cells. A yeast two-hybrid system-based screen was devised to identify randomly mutagenized FANCE proteins capable of interaction with FANCC but not with FANCD2. Exogenous expression of these mutants in an FA-E cell line and subsequent evaluation of FANCD2 monoubiquitination and DNA cross-linker sensitivity indicated a critical role for the FANCE/FANCD2 interaction in maintaining FA pathway integrity. Three-hybrid experiments also demonstrated the ability of FANCE to mediate the interaction between FA core complex components FANCC and FANCF, indicating an additional role for FANCE in complex assembly. Thus, FANCE is shown to be a key mediator of protein interactions both in the architecture of the FA protein complex and in the connection of complex components to the putative downstream targets of complex activity.

Fanconi anemia protein complex: mapping protein interactions in the yeast 2- and 3-hybrid systems.

Fanconi anemia (FA) is an autosomal recessive syndrome characterized by progressive bone marrow failure and cancer predisposition. Eight FA complementation groups have been identified. The FANCA, FANCC, FANCE, FANCF, and FANCG proteins form a nuclear complex required for the monoubiquination of the FANCD2 protein. To investigate the architecture of the FA protein complex, the yeast 2-hybrid system was used to map contact points of the FANCA/FANCG, FANCC/FANCE, and FANCF/FANCG interactions. FANCG was shown to interact with both the amino-terminus of FANCA and the carboxyl-terminal region of FANCF. A FANCG mutant truncated at the carboxyl-terminus retained the ability to interact with FANCA. The interaction between FANCG and FANCF was ablated by a Leu71Pro mutant of FANCG. A central region of FANCE was sufficient for FANCC binding. A Leu554Pro mutant of FANCC failed to interact with FANCE. To further examine complex assembly, the yeast 3-hybrid system was used to investigate the ability of FANCG to act as a molecular bridge in mediating interaction between other FA proteins. FANCG was able to mediate interaction between FANCA and FANCF, as well as between monomers of FANCA. Direct interaction between FANCE and FANCD2 was also demonstrated in the yeast 2-hybrid system. This interaction involving an amino-terminal region of FANCD2 may provide a link between the FA protein complex and its downstream targets.

Cloning and mutation analysis of ZFP276 as a candidate tumor suppressor in breast cancer.

Loss of heterozygosity (LOH) involving chromosome 16q23.4 occurs frequently in breast tumors, which suggests that this region may contain a tumor suppressor gene. Since ZFP276 is located in this region, we have therefore cloned and performed mutation analysis of its coding region in 70 breast tumors. One silent polymorphism and two alterations predicted to result in amino acid changes were detected. Absence of the wild-type allele in tumors carrying the E530D variant suggests a possible role for this change in tumorigenesis.

Targeted disruption of exons 1 to 6 of the Fanconi Anemia group A gene leads to growth retardation, strain-specific microphthalmia, meiotic defects and primordial germ cell hypoplasia.

Fanconi Anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents. Recent studies suggest that FA proteins share a common pathway with BRCA proteins. To study the in vivo role of the FA group A gene (Fanca), gene-targeting techniques were used to generate Fanca(tm1Hsc) mice in which Fanca exons 1-6 were replaced by a beta-galactosidase reporter construct. Fanca(tm1.1Hsc) mice were generated by Cre-mediated removal of the neomycin cassette in Fanca(tm1Hsc) mice. Fanca(tm1.1Hsc) homozygotes display FA-like phenotypes including growth retardation, microphthalmia and craniofacial malformations that are not found in other Fanca mouse models, and the genetic background affects manifestation of certain phenotypes. Both male and female mice homozygous for Fanca mutation exhibit hypogonadism, and homozygous females demonstrate premature reproductive senescence and an increased incidence of ovarian cysts. We showed that fertility defects in Fanca(tm1.1Hsc) homozygotes might be related to a diminished population of primordial germ cells (PGCs) during migration into the gonadal ridges. We also found a high level of Fanca expression in pachytene spermatocytes. Fanca(tm1Hsc) homozygous males exhibited an elevated frequency of mispaired meiotic chromosomes and increased apoptosis in germ cells, implicating a role for Fanca in meiotic recombination. However, the localization of Rad51, Brca1, Fancd2 and Mlh1 appeared normal on Fanca(tm1Hsc) homozygous meiotic chromosomes. Taken together, our results suggest that the FA pathway plays a role in the maintenance of reproductive germ cells and in meiotic recombination.

Short-term granulocyte colony-stimulating factor and erythropoietin treatment enhances hematopoiesis and survival in the mitomycin C-conditioned Fancc(-/-) mouse model, while long-term treatment is ineffective.

Transient treatment with cytokines appears to improve hematopoietic function in Fanconi anemia; however, the effectiveness or adverse effect of long-term treatment is not known. The mitomycin C-treated Fancc(-/-) mouse provides a valuable model to address long-term efficacy of such treatment. Fancc(-/-) mice injected with granulocyte colony-stimulating factor, erythropoietin, or both cytokines showed a delay in mitomycin C (MMC)-induced bone marrow (BM) failure compared to untreated mice. However, long-term cytokine exposure followed by MMC challenges did not protect mice from the reduction of peripheral blood counts or the number of early myeloid progenitors. These results suggest that cytokine treatment may be beneficial only in the short-term, while long-term treatment is not protective for BM aplasia.

Transgenic expression in mouse lung reveals distinct biological roles for the adenovirus type 5 E1A 243- and 289-amino-acid proteins.

Little is known about the biological significance of human adenovirus type 5 (Ad5) E1A in vivo. However, Ad5 E1A is well defined in vitro and can be detected frequently in the lungs of patients with pulmonary disease. Transgenic expression of the Ad5 E1A gene targeted to the mouse lung reveals distinct biological effects caused by two Ad5 E1A products. Either of two Ad5 E1A proteins was preferentially expressed in vivo in the transgenic lungs. The preferential expression of the Ad5 E1A 243-amino-acid (aa) protein at a moderate level was associated with cellular hyperplasia, nodular lesions of proliferating lymphocyte-like cells, and a low level of p53-dependent apoptosis in the lungs of transgenic mice. In contrast, the preferential expression of the Ad5 E1A 289-aa protein at a moderate level resulted in a proapoptotic injury and an acute pulmonary proinflammation in the lungs of transgenic mice, mediated by multiple apoptotic pathways, as well as an enhancement of the host immune cell response. Expression of the Ad5 E1A 243-aa protein resulted in proliferation-stimulated p53 upregulation, while expression of the Ad5 E1A 289-aa protein led to DNA damage-induced p53 activation. These data suggest that the Ad5 E1A 243- and 289-aa proteins lead to distinct biological roles in vivo.

Regulation of the Fanconi anemia group C protein through proteolytic modification.

The function of the Fanconi anemia group C protein (FANCC) is still unknown, though many studies point to a role in damage response signaling. Unlike other known FA proteins, FANCC is mainly localized to the cytoplasm and is thought to act as a messenger of cellular damage rather than an effector of repair. FANCC has been shown to interact with several cytoplasmic and nuclear proteins and to delay the onset of apoptosis through redox regulation of GSTP1. We investigated the fate and function of FANCC during apoptosis. Here we show that FANCC undergoes proteolytic modification by a caspase into a predominant 47-kDa ubiquitinated protein fragment. Lack of proteolytic modification at the putative cleavage site delays apoptosis but does not affect MMC complementation. These results suggest that FANCC function is regulated through proteolytic processing.