Analysis of cancer gene functions through gene inhibition with antisense oligonucleotides.
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Antineoplastic Agents↗
Biomedical subjects
J T Holt
Publications and source records attributed to J T Holt.
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 BRCA2 functions 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 of BRCA1 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 without BRCA1 showed decreased TCR and radiosensitivity. These findings give physiologic significance to the interaction of BRCA1 with the basal transcription machinery.
BRCA1 Protein↗
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 activating ERK1 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.
3T3 Cells↗
Ovarian cancer BRCA1 gene therapy: Phase I and II trial differences in immune response and vector stability.
Gene therapy with viral vectors has shown some promise in nude mice models and in initial Phase I trials of patients with extensive metastatic cancer. A Phase I clinical trial (D. L. Tait et al., Clin. Cancer Res., 3: 1959-1968, 1997) of ovarian cancer patients treated with i.p. retroviral LXSN-BRCA1sv gene therapy reported stable vector, minimal antibody response, and tumor reduction. We initiated a Phase II trial on patients with less extensive disease to evaluate vector pharmacokinetics, immune response, toxicity, and efficacy. Patients received a surgically implanted peritoneal catheter to administer infusions of vector, as well as to retrieve daily samples of peritoneal fluid for analysis. Ovarian cancer patients received four daily i.p. injections of LXSN-BRCA1sv vector therapy for three cycles, 4 weeks apart. Patient peritoneal fluid and plasma were analyzed extensively by PCR, Western blot, complement level (CH50), and chemical and hematological tests. Phase II patients showed no response, no disease stabilization, and little or no vector stability. Because of vector instability and rapid antibody development, which differed dramatically from the Phase I trial data, the trial was terminated after treatment of six patients. Immune system status appears to have played a major role in whether gene therapy was effective. Comparison of Phase I and II patients showed significant differences in tumor burden, immune system status, and response to BRCA1 gene therapy.
Adolescent↗
Double-strand break repair deficiency and radiation sensitivity in BRCA2 mutant cancer cells.
BACKGROUND: The protein product of the BRCA2 gene mediates repair of double-strand breaks in DNA. Because a number of cancer therapies exert cytotoxic effects via the initiation of double-strand breaks, cancers comprised of cells carrying BRCA2 gene mutations may be more amenable to treatment with agents that cause such breaks. METHODS: We identified a human pancreatic adenocarcinoma cell line lacking one copy of the BRCA2 gene and containing a mutation (6174delT) in the remaining copy. In vitro and in vivo experiments were conducted with this cell line and with other carcinoma cell lines matched for similar genetic mutations, similar differentiation status, and/or similar carcinoma type to examine double-strand break repair, sensitivity to drugs that induce double-strand breaks, and radiation sensitivity. RESULTS: BRCA2-defective cells were unable to repair the double-strand DNA breaks induced by ionizing radiation. These cells were also markedly sensitive to mitoxantrone, amsacrine, and etoposide (drugs that induce double-strand breaks) (two-sided P = .002) and to ionizing radiation (two-sided P = .001). Introduction of antisense BRCA2 deoxyribonucleotides into cells possessing normal BRCA2 function led to increased sensitivity to mitoxantrone (two-sided P = .008). Tumors formed by injection of BRCA2-defective cells into nude mice were highly sensitive (>90% tumor size reduction, two-sided P = .002) to both ionizing radiation and mitoxantrone when compared with tumors exhibiting normal BRCA2 function. Histologic analysis of irradiated BRCA2-defective tumors showed a large degree of necrosis compared with that observed for control tumors possessing normal BRCA2 function. CONCLUSION: BRCA2-defective cancer cells are highly sensitive to agents that cause double-strand breaks in DNA.
Adenocarcinoma↗
Antisense c-myc retroviral vector suppresses established human prostate cancer.
Prostate cancer eventually becomes androgen resistant, resumes growth, and kills the patient. Characterization of genetic events that lead to androgen refractory prostatic neoplasia has revealed the frequent overexpression of c-myc and uncontrolled prostate cancer proliferation. A novel strategy to combat advanced prostate cancer utilized a replication incompetent retrovirus that contained the mouse mammary tumor virus (MMTV) promoter within the retroviral vector to allow transcription of antisense c-myc gene within target prostate tumor cells. The transduction of cultured DU145 cells by XM6:MMTV-antisense c-myc RNA retrovirus did not affect cell proliferation in culture, yet a single direct injection of MMTV-antisense c-myc viral media into established DU145 tumors in nude mice produced a 94.5% reduction in tumor size compared to tumors treated with control virus MTMV sense fos and untreated tumor by 70 days. Two animals in the antisense c-myc-treated group had complete regression of their tumors. Histopathological examination of the tumors revealed that MMTV-antisense c-myc-transduced DU145 tumors had increased tumor cell differentiation, decreased invasion, and a marked stromal response. The mechanism for the antitumor effect of MMTV-antisense c-myc retrovirus appears to be suppression of c-myc mRNA and protein, and decreased bcl-2 protein. The in vivo transduction of prostate cancer cells with MMTV-antisense c-myc retroviruses reduced tumor growth by suppressing c-myc, resulting in the down-regulation of bcl-2 protein. Consequently, the MMTV-antisense c-myc retrovirus may be useful for gene therapy against advanced, hormone-refractory prostate cancer.
Animals↗
Ovarian cancer gene therapy.
Retroviral-mediated delivery of BRCA1 gene therapy (LXN-BRCA1sv, a normal splice variant form of BRCA1) was tested extensively in mouse models. It was found to be effective in reducing tumor burden and to be minimally toxic. Twelve phase I clinical trial patients with recurrent or persistent epithelial ovarian cancer were treated with one to three cycles of intraperitoneal vector. There was minimal toxicity, four patients developed fevers (< 102.5 degrees F) and three had sterile peritonitis, which resolved within 48 hours. The vector was found to be fairly stable in some patients at 24 hours as well as transferred into and expressed in patient tissues. Stable disease was noticed in 8 of the 12 patients, suggesting that the peritoneal cavity may be an appropriate site for gene therapy.
Animals↗
Breast cancer genes: therapeutic strategies.
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.
Animals↗
Finkel-Biskis-Reilly osteosarcoma virus v-Fos inhibits adipogenesis and both the activity and expression of CCAAT/enhancer binding protein alpha, 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/EBPalpha. 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)-alpha expression. These data implicate C/EBPalpha as a protein involved in the generation of liposarcomas.
3T3 Cells↗
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.
Animals↗
RAP-PCR using RNA from tissue microdissection.
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Base Sequence↗
A phase I trial of retroviral BRCA1sv gene therapy in ovarian cancer.
Gene transfer of BRCA1sv (a normal splice variant of BRCA1) into ovarian cancer cells produces growth inhibition in vitro and tumor suppression in nude mouse xenografts. As an initial step toward gene replacement therapy for ovarian cancer, we conducted a Phase I trial to assess the pharmacokinetics and toxicity of i.p. BRCA1sv retroviral vector therapy. Following placement of an indwelling Port-a-Cath in patients, a dose escalation study was performed of four daily i.p. infusions spanning doses from 3 to 300 ml (i.e., 10(10) viral particles) at half-log intervals (23 cycles in 12 patients). Gene transfer and expression were documented by PCR, Southern blot, reverse transcription-PCR, and nuclease protection assays. Pharmacokinetics were assessed by PCR and Southern blots detecting vector DNA, and toxicity was evaluated by clinical exam and fluid analysis. Three of 12 patients developed an acute sterile peritonitis, which spontaneously resolved within 48 h. Plasma and peritoneal antibodies to the retroviral envelope protein were detected only in patients treated with the highest dose levels but not in others, despite repeat dosing for an interval of up to 4 months. Eight patients showed stable disease for 4-16 weeks, and three patients showed tumor reduction with diminished miliary tumor implants at reoperation (two patients) and radiographic shrinkage of measurable disease (one patient). The vector-related complication of peritonitis was observed in three patients but resolved quickly as in preclinical mouse studies. Ovarian cancer may provide an important model for retroviral gene therapy studies due to vector stability, minimal antibody response, and access to tumor by i.p. therapy.
Adult↗
Gene therapy for the treatment of metastatic breast cancer by in vivo transduction with breast-targeted retroviral vector expressing antisense c-fos RNA.
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Adolescent↗
Tissue-targeted antisense c-fos retroviral vector inhibits established breast cancer xenografts in nude mice.
The c-fos proto-oncogene has been implicated as a regulator of estrogen-mediated cell proliferation. We have tested the tissue specificity and antitumor efficacy of a mouse mammary tumor virus-regulated antisense c-fos retroviral vector. Systemically administered vector could be detected in several tissues but was only expressed in breast epithelium, thus supporting targeting to mouse mammary tumor virus-regulated tissues. Ex vivo transduction of 30-70% of MCF-7 human breast cancer cells produced expression of antifos RNA, decreased expression of the c-fos target mRNA, induction of differentiation, and inhibition of s.c. tumor growth and invasiveness. In vivo transduction of established i.p. MCF-7 tumors with a single injection of XM6:antifos inhibited tumor growth in athymic mice with a corresponding inhibition of c-fos, transforming growth factor beta1 and transforming growth factor alpha expression. Four daily injections with the antifos RNA induced a much larger MCF-7 i.p. tumor inhibition, with a marked prolongation of survival in the absence of any host tissue toxicity. These results indicate that inhibition of key nuclear genes such as c-fos may lead to disruption of paracrine factors and an antitumor effect, providing a strategy for cancer gene therapy.
Animals↗
Growth retardation and tumour inhibition by BRCA1.
Inherited mutations in BRCA1 predispose to breast and ovarian cancer, but the role of BRCA1 in sporadic breast and ovarian cancer has previously been elusive. Here, we show that retroviral transfer of the wild-type BRCA1 gene inhibits growth in vitro of all breast and ovarian cancer cell lines tested, but not colon or lung cancer cells or fibroblasts. Mutant BRCA1 has no effect on growth of breast cancer cells; ovarian cancer cell growth is not affected by BRCA1 mutations in the 5' portion of the gene, but is inhibited by 3' BRCA1 mutations. Development of MCF-7 tumours in nude mice is inhibited when MCF-7 cells are transfected with wild-type, but not mutant, BRCA1. Most importantly, among mice with established MCF-7 tumours, peritoneal treatment with a retroviral vector expressing wild-type BRCA1 significantly inhibits tumour growth and increased survival.
Animals↗
BRCA1 is secreted and exhibits properties of a granin.
Germline mutations in BRCA1 are responsible for most cases of inherited breast and ovarian cancer. However, the function of the BRCA1 protein has remained elusive. We now show that BRCA1 encodes a 190-kD protein with sequence homology and biochemical analogy to the granin protein family. Interestingly, BRCA2 also includes a motif similar to the granin consensus at the C terminus of the protein. Both BRCA1 and the granins localize to secretory vesicles, are secreted by a regulated pathway, are post-translationally glycosylated and are responsive to hormones. As a regulated secretory protein, BRCA1 appears to function by a mechanism not previously described for tumour suppressor gene products.
Amino Acid Sequence↗
Myristylation of FBR v-fos dictates the differentiation pathways in malignant osteosarcoma.
Myristylation of FBR v-fos, a c-fos retroviral homologue that causes osteosarcomas in mice, determines many of its transcriptional properties in vitro. To determine whether myristylation of FBR v-fos contributes to in vivo tumorigenicity, we examined its transforming capability in comparison to a nonmyristylated FBR v-fos (G2A-R). Retroviral infections with FBR v-fos and G2A-R transform BALB/c-3T3 cells. The number, size, and cellular morphology of foci generated by both FBR and G2A-R are indistinguishable. However, marked biological differences were found in transgenic mice expressing either the myristylated FBR v-fos or the nonmyristylated G2A-R. 11 of 26 FBR v-fos transgenic mice died as a result of gross tumor burden. None of the 28 G2A-R transgenic mice died from tumor burden, and only two of the G2A-R mice developed bone tumors. Histologic examination of the tumors reveals that the FBR v-fos bone tumors contain malignant cells with features of four cell lineages (osteocytes, chondrocytes, myocytes, and adipocytes) in an environment rich in extracellular matrix (ECM). However, the G2A-R tumors exist in an environment devoid of ECM and display malignant cells with features of adipocytes. Masson staining reveals that the ECM of the FBR tumors stains strongly for collagen. Immunohistochemical staining with collagen III antibody demonstrates an abundance of collagen III expression in this ECM. While NH2-terminal myristylation is not required for FBR immortalization and transformation, it is essential in determining the degree of differentiation and tumorigenicity of malignant cells.
3T3 Cells↗
Antisense techniques.
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Adenosine Triphosphate↗