Jeffrey T. Holt

Development of mammary hyperplasia and neoplasia in MMTV-TGFα transgenic mice

Abstract To study the role of transforming growth factor α (TGFα) in normal mammary development and mammary neoplasia in vivo, we have generated transgenic mice in which a human TGFα cDNA is expressed under the control of the MMTV enhancer/promoter. Overexpression of TGFα in the mammary epithelium, as confirmed by in situ hybridization and immunohistochemistry, is associated with hyperplasia of alveoli and terminal ducts in virgin female and pregnant transgenic mice. A range of morphologic abnormalities including lobular hyperplasia, cystic hyperplasia, adenoma, and adenocarcinoma is seen in mammary tissue of transgenic females. In contrast, no morphologic abnormalities are seen in transgenic males in spite of TGFα overexpression in salivary glands and reproductive organs. TGFα can therefore act as an oncogene in vivo and appears to predispose mammary epithelium to neoplasia and carcinoma.

BRCA1 Expression Restores Radiation Resistance in BRCA1-defective Cancer Cells through Enhancement of Transcription-coupled DNA Repair

The breast cancer predisposition genes,BRCA1 and BRCA2, are responsible for the vast majority of hereditary breast cancer. Although BRCA2functions to help the cell repair double-stranded DNA breaks, the function of BRCA1 remains enigmatic. Here, we develop a human genetic system to study the role of BRCA1 in oxidative DNA damage. We show that human cancer cells containing mutated BRCA1 are hypersensitive to ionizing radiation. This hypersensitivity can be reversed by the expression of forms ofBRCA1 that are not growth suppressing. Reversal of hypersensitivity requires the ring finger of BRCA1, its transactivation domain, and its BRCT domain. Lastly, we show that unlike BRCA2, BRCA1 does not function in the repair of double-stranded DNA breaks. Instead, it functions in transcription-coupled DNA repair (TCR). TCR ability correlated with radioresistance as cells containing BRCA1 showed both increased TCR and radioresistance, whereas cells withoutBRCA1 showed decreased TCR and radiosensitivity. These findings give physiologic significance to the interaction ofBRCA1 with the basal transcription machinery.

Mitogen-activated Protein Kinase Kinase 2 Activation Is Essential for Progression through the G2/M Checkpoint Arrest in Cells Exposed to Ionizing Radiation

An increasing body of evidence suggests that mitogen-induced activation of the RAF/ERK signaling pathway is functionally separate from the stress-induced activation of the SEK/JNK/p38 signaling pathway. In general, stress stimuli strongly activate the p38s and the JNKs while only weakly activatingERK1 and ERK2. However, a number of independent groups have now shown that the RAF/ERK signaling pathway is strongly activated by ionizing radiation. In this work, we examine this paradox. We show that both mitogen-activated protein (MAP) kinase kinase 1 (MEK1) and MAP kinase kinase 2 (MEK2) are activated by ionizing radiation. Blockage of this activation through the use of dominant negative MEK2 increases sensitivity of the cell to ionizing radiation and decreases the ability of a cell to recover from the G2/M cell cycle checkpoint arrest. Blocking MEK2 activation does not affect double-strand DNA break repair, however. Although MEK1 is activated to a lesser extent by ionizing radiation, expression of a dominant negative MEK1 does not affect radiation sensitivity of the cell, the G2/M checkpoint of the cell, or double-strand break repair. Because ionizing radiation leads to a different cell cycle arrest (G2/M arrest) than that typically seen with other stress stimuli, and because we have shown that MEK2 can affect G2/M checkpoint kinetics, these results provide an explanation for the observation that the MEKs can be strongly activated by ionizing radiation and only weakly activated by other stressful stimuli.

Redox-sensitive interaction between KIAA0132 and Nrf2 mediates indomethacin-induced expression of γ-glutamylcysteine synthetase

Exposure of HepG2 cells to nonsteroidal anti-inflammatory drugs (i.e., indomethacin and ibuprofen; NSAIDs) as well as resveratrol, caused increased expression of the mRNAs coding for the catalytic (Gclc) and modifier (Gclm) subunits of the glutathione synthetic enzyme, gamma-glutamylcysteine synthetase. In addition, indomethacin exposure increased intracellular glutathione content as well as inhibited glutathione depletion and cytotoxicity caused by diethyl maleate. Indomethacin-induced increases in the expression of gamma-glutamylcysteine synthetase mRNA were preceded by increases in steady state levels of intracellular pro-oxidants and glutathione disulfide accumulation. Simultaneous incubation with the thiol antioxidant N-acetylcysteine (NAC) inhibited indomethacin-mediated increases in GCLC mRNA, suggesting that increases in GCLC message were triggered by changes in intracellular oxidation/reduction (redox) reactions. Indirect immunofluorescence using intact cells demonstrated that indomethacin induced the nuclear translocation of Nrf2, a transcription factor believed to regulate GCLC expression. Immunoprecipitation studies showed that indomethacin treatment also inhibited Nrf2 tethering to KIAA0132 (the human homolog of Keap1 accession #D50922), which is believed to be a negative regulator of Nrf2. Consistent with this idea, over-expression of Nrf2 increased GCLC reporter gene expression and over-expression of KIAA0132 inhibited GCLC reporter gene activity as well as inhibited indomethacin-induced increases in the expression of GCLC. Finally, simultaneous treatment with NAC inhibited both indomethacin-induced release of Nrf2 from KIAA0132 and indomethacin-induced nuclear translocation of Nrf2. These results demonstrate that NSAIDs and resveratrol cause increases in the expression of gamma-glutamylcysteine synthetase mRNA and identify these agents as being capable of stimulating glutathione metabolism. These results also support the hypothesis that indomethacin-induced transcriptional activation of GCLC involves the redox-dependent release of KIAA0132 from Nrf2 followed by the nuclear translocation of Nrf2.

Rad51 overexpression rescues radiation resistance in BRCA2-defective cancer cells.

Breast cancers with BRCA2 mutations exhibit DNA repair defects and are particularly sensitive to radiation. BRCA2 interacts with Rad51 in a complex manner involving internal BRC and C‐terminal TR2 domains which play a key role in homologous recombination. BRCA2 expression also modulates Rad51 protein levels such that Rad51 protein is relatively decreased in BRCA2‐defective cancer cells. This is mediated in part through BRCA2s capacity to protect Rad51 from caspase‐3 proteolytic degradation. In order to distinguish between functional and expression related roles for BRCA2 we studied the results of Rad51 overexpression in mouse and human cells with inactivating BRCA2 mutations. The results show that overexpression of wild‐type Rad51 partially rescues BRCA2 deficiency but that overexpression of a caspase‐3 resistant Rad51 completely complements the BRCA2 defect in radiation responsiveness. These results indicate that Rad51 can compensate for some aspects of a BRCA2 gene defect and suggest that Rad51 expression levels may be an important modifier of the BRCA2 defective genotype.

Finkel-Biskis-Reilly mouse osteosarcoma virus v-fos inhibits the cellular response to ionizing radiation in a myristoylation-dependent manner.

DNA damage is recognized as a central component of carcinogenesis. DNA-damaging agents activate a number of signal transduction pathways that lead to repair of the DNA, apoptosis, or cell cycle arrest. It is reasoned that a cell deficient in DNA repair is more likely to acquire other cancer-promoting mutations. Despite the recent interest in the link between DNA damage and carcinogenesis, retroviral oncogenes have not yet been shown to affect the DNA damage-signaling pathway. In this report, we show that Finkel-Biskis-Reilly mouse osteosarcoma virus (FBR) v-fos, the retroviral homologue of the c-fos proto-oncogene, inhibits the cellular response to ionizing radiation. Cells that express FBR v-Fos show a decreased ability to repair DNA damage caused by ionizing radiation, and these cells show decreased survival in response to ionizing radiation. In addition, FBR v-Fos inhibits DNA-dependent protein kinase, a kinase specifically activated upon exposure to ionizing radiation. These effects were specific to ionizing radiation, as no effect of FBR v-Fos on the UV light signaling pathway was seen. Last, these effects were dependent on a lipid modification required for FBR v-Fos tumorigenesis, that of myristoylation of FBR v-Fos. A non-myristoylated mutant FBR v-Fos caused none of these effects. This study suggests that a retroviral oncogene can lead to an increased genomic instability, which can ultimately increase the carcinogenic potential of a cell.

[23] Antisense techniques

Publisher Summary This chapter focuses on the strategies and controls that can limit artifactual results: in particular, the use of reversal or rescue methods to hybrid arrest the antisense effect. The ability to manipulate gene expression within mammalian cells has provided powerful experimental approaches for studies of gene function and gene regulation. Methods that inhibit gene expression are particularly important because they permit studies probing the normal function of a specific gene product within a cell. Antibody injection and antisense studies have shown that cellular protooncogenes, particularly transcription factor protooncogenes, have important roles regulating proliferation and differentiation. The antisense methodologies produce inhibition of specific gene products by exploiting hybridization of complementary nucleic acids, resulting in decreased mRNA stability, or through a block in mRNA processing, transport, or translation. Transfection of antisense plasmids generally requires the isolation of stable transformants that can produce artifactual results due to biased selection pressures and direct effects of inducing agents on experimental systems. The addition of antisense oligonucleotides can occasionally generate artifactual results due to toxicity and not as a direct consequence of hybridization and consequent antisense inhibition. Major considerations for design of antisense oligonucleotide include: (1) site of target sequence within gene, (2) length of antisense oligonucleotide, and (3) secondary structure of antisense oligonucleotide.

Gene therapy for carcinoma of the breast: Therapeutic genetic correction strategies

Gene therapy is a therapeutic approach that is designed to correct specific molecular defects that contribute to the cause or progression of cancer. Genes that are mutated or deleted in cancers include the cancer susceptibility genes p53 and BRCA1. Because mutational inactivation of gene function is specific to tumor cells in these settings, cancer gene correction strategies may provide an opportunity for selective targeting without significant toxicity for normal nontumor cells. Both p53 and BRCA1 appear to inhibit cancer cells that lack mutations in these genes, suggesting that the so-called gene correction strategies may have broader potential than initially believed. Increasing knowledge of cancer genetics has identified these and other genes as potential targets for gene replacement therapy. Initial patient trials of p53 and BRCA1 gene therapy have provided some indications of potential efficacy, but have also identified areas of basic and clinical research that are needed before these approaches may be widely used in patient care.

Finkel-Biskis-Reilly Osteosarcoma Virus v-Fos Inhibits Adipogenesis and Both the Activity and Expression of CCAAT/Enhancer Binding Protein α, a Key Regulator of Adipocyte Differentiation

Finkel-Biskis-Reilly (FBR) osteosarcoma virus v-Fos causes tumors of mesenchymal origin, including osteosarcomas, rhabdomyosarcomas, chondrosarcomas, and liposarcomas. Because the cell of origin in all these tumors is a pluripotent mesenchymal cell, the variety of tumors seen in mice which express FBR v-Fos implies that FBR v-Fos inhibits multiple differentiation pathways. To study the mechanism of FBR v-Fos’ inhibition of mesenchymal differentiation, we utilized an in vitro model of adipocyte differentiation. We show by both morphological and biochemical means that FBR v-Fos inhibits adipocyte differentiation in vitro. This inhibition is due to FBR v-Fos’ inhibition of the growth arrest characteristic of terminal differentiation and FBR v-Fos’ inhibition of the expression and activity of a key regulator of this growth arrest, C/EBPα. The in vitro inhibition of adipogenesis by FBR v-Fos has in vivo significance as immunostaining of FBR v-Fos-induced tumors shows no CCAAT/enhancer binding protein (EBP)-α expression. These data implicate C/EBPα as a protein involved in the generation of liposarcomas.

Breast cancer genes: therapeutic strategies.

ABSTRACT: Although effective treatments for breast cancer predated the identification of causative molecular defects in humans, it is widely hoped that an understanding and/or manipulation of the key genetic events will lead to even more effective therapies or even cures. Powerful methods of positional cloning and gene identification have identified the breast cancer genes, BRCA1 and BRCA2, which together are responsible for the majority of cases of hereditary breast and ovarian cancer. Although the BRCA1 gene is rarely mutated in sporadic breast or ovarian cancer, levels of BRCA1 mRNA and protein are markedly decreased in the majority of sporadic cases of cancer. This suggests that hereditary and sporadic breast cancer share common genetic themes and that treatments aimed at increasing levels of BRCA1 or BRCA2 may be useful for both hereditary and sporadic cancers. We have demonstrated that gene transfer of wild‐type BRCA1 inhibits the growth of sporadic breast and ovarian cancer cells and suppresses growth of established breast and ovarian tumor models in nude mice. Mutant BRCA1 genes do not inhibit growth or suppress tumor, providing additional evidence that BRCA1 is a tumor‐suppressor gene. Strategies designed to increase BRCA1 expression or development of BRCA1‐mimetic agents may be ultimately useful as therapeutic approaches.