Antisense ATM gene therapy: a strategy to increase the radiosensitivity of human tumors.

Atm, the gene mutated in ataxia-telangiectasia (AT) patients, is an essential component of the signal transduction pathway that responds to DNA damage due to ionizing radiation (IR). We attenuated ATM protein expression in human glioblastoma cells by expressing antisense RNA to a functional domain of the atm gene. While ATM expression decreased, constitutive expression of p53 and p21 increased. Irradiated ATM-attenuated cells failed to induce p53, demonstrated radioresistant DNA synthesis, and increased radiosensitivity. Antisense-ATM gene therapy in conjunction with radiation therapy may provide a novel strategy for the treatment of cancer.

Paclitaxel sensitivity correlates with p53 status and DNA fragmentation, but not G2/M accumulation.

PURPOSE: The antitumor agent paclitaxel (Taxol) has been shown to arrest cells in mitosis through microtubule stabilization and to induce apoptosis. The tumor suppressor gene p53 is implicated in the regulation of cell cycle checkpoints and can mediate apoptotic cell death. Although initial studies demonstrated that various DNA-damaging agents can induce p53, more recent studies have also shown p53 induction following nonDNA-damaging agents, including paclitaxel. We investigated the influence of p53 abrogation on paclitaxel-induced cell kill and correlated the extent of mitotic arrest and DNA fragmentation by paclitaxel with the drug's cytotoxic effect. MATERIALS AND METHODS: The parental human colorectal carcinoma cell line (RKO) with wild-type p53 alleles, and two transfected RKO cell lines with inactivated p53 (RKO.p53.13 with transfected mutant p53 and RC 10.3 with HPV-16-derived E6 gene) were exposed to graded doses of paclitaxel (1-100 nM) for 24-h intervals. The functional status of p53 in cells was assessed by thymidine and BrdU incorporation following exposure to ionizing radiation (4 Gy). Reproductive integrity following paclitaxel treatment was assessed by clonogenic assay. Immunolabeling and microscopic evaluation were used to assess mitotic accumulation and micronucleation. Apoptosis was assayed using DNA fragmentation analyses. RESULTS: A 4-fold increase in paclitaxel sensitivity was observed among RKO cells deficient in p53 function compared with wild-type RKO cells (IC 50: 4 nM, 1 nM, 1nM for RKO, RKO.p53.13, RC 10.3, respectively). The increased cytotoxic effect in RKO cells with inactive p53 correlated with an increased propensity towards micronucleation and DNA fragmentation following paclitaxel treatment. However, no significant difference in peak mitotic accumulation was observed among RKO cells with functional or abrogated p53. CONCLUSIONS: RKO cells lacking functional p53 demonstrate significantly enhanced sensitivity to paclitaxel compared with that of wild-type RKO cells. This response corresponded with increased micronucleation and DNA fragmentation in cells deficient in p53 function. Although previous published reports of enhanced paclitaxel sensitivity in p53-deficient cells correlated this finding with increased G2/M arrest, we did not observe any significant correlation between paclitaxel-induced cell kill and the degree of mitotic arrest. Our data suggest that apoptosis is the predominant mechanism of paclitaxel cytotoxicity in RKO cells and is likely mediated by a p53-independent process.

Penclomedine-induced DNA fragmentation and p53 accumulation correlate with reproductive cell death in colorectal carcinoma cells with altered p53 status.

Penclomedine, a synthetic pyridine derivative, has documented antitumor activity and is being investigated in clinical trials. Its mechanism of action is unknown although it may be metabolized to a free radical, DNA-reactive species. We previously reported that telomerase positive colorectal carcinoma (RKO) cells with abrogated p53 function were more sensitive to penclomedine than were telomerase positive cells with wild-type p53. The present study demonstrates that significant differences in DNA fragmentation in response to penclomedine were observed in RKO cells lacking functional p53 compared with RKO cells with normal p53 function. No differences in DNA fragmentation in response to ionizing radiation were seen in RKO cells with normal or abrogated p53 function. RKO cells with functional p53 respond to penclomedine treatment with a dose-dependent increase in p53 protein levels. However, RKO cells with abrogated p53 function did not show any such change in p53 protein levels. Further, p53-independent increase of p21 was observed, although the significance of this response remains uncertain. These studies suggest that penclomedine may have a therapeutic advantage in killing cells that have abrogated p53 function.

Molecular cloning and chromosomal localization of Chinese hamster telomeric protein chTRF1. Its potential role in chromosomal instability.

Chinese hamster cells frequently have altered karyotypes. To investigate the basis of recent observations that karyotypic alterations are related to telomeric fusions, we asked whether these alterations are due to lack of telomere repeat binding factor/s. Further, Chinese hamster chromosomes contain large blocks of interstitial telomeric repeats, which are preferentially involved in chromosome breakage and exchange, rendering it an interesting model for such studies. Here, we report on the cloning and the chromosomal localization of the Chinese hamster telomere repeat binding factor, chTRF1. The sequence analysis revealed, similar to human TRF1 (hTRF1), an N-terminal acidic domain, a TRF1 specific DNA binding motif and a C-terminal Myb type domain. Unlike mouse TRF1 (mTRF1), chTRF1 shows 97.5% identity to hTRF1. chTRF1 gene was localized on the long arm of chromosome 5. In vitro translation of chTRF1 resulted in protein product similar in molecular weight to hTRF1. Immunostaining of Chinese hamster ovary cells (CHO) with anti-TRF1 antibody revealed punctate nuclear staining. At metaphase, antibodies failed to detect TRF1 on most of the chromosome ends and the interstitial telomeric repeat bands. These studies suggest that chTRF1 does not bind the interstitial telomeric repeats, and its presence at the metaphase chromosome ends is limited. The later could be a factor contributing to frequent karyotypic alterations observed in Chinese hamster cells.

Influence of ATM function on telomere metabolism.

The ATM gene product, which is defective in the cancer-prone disorder ataxia telangiectasia, has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination and cell cycle control. The ATM gene has homology with the TEL1 gene of yeast, mutations of which lead to shortened telomeres. To test the hypothesis that the ATM gene product is involved in telomere metabolism, we examined telomeric associations (TA), telomere length, and telomerase activity in human cells expressing either dominant-negative or complementing fragments of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper (LZ) motif mimics that of ataxia telangiectasia (A-T) cells. These transfected RKO cells relative to transfected controls had a higher frequency of cells with TA and shortened telomeres, but no detectable change in telomerase activity. In addition, the percentage of cells with TA after gamma irradiation was higher in the transfected RKO cells with dominant negative activity of the ATM gene, compared to control cells. SV40 transformed fibroblasts derived from an A-T patient and transfected with a complementing carboxyl terminal kinase region of the ATM gene had a reduced frequency of cells with TA, with no effect on the telomere length or telomerase activity. The present studies using isogenic cells with manipulated ATM function demonstrate a role for the ATM gene product in telomere metabolism.

Induction of cyclophosphamide-resistance by aldehyde-dehydrogenase gene transfer.

The identification of genes inducing resistance to anticancer chemotherapeutic agents and their introduction into hematopoietic cells represents a promising approach to overcome bone marrow toxicity, the limiting factor for most high-dose chemotherapy regimens. Because resistance to cyclophosphamide has been correlated with increased levels of expression of the aldehyde-dehydrogenase (ALDH1) gene in tumor cell lines in vitro, we tested whether ALDH1 overexpression could directly induce cyclophosphamide resistance. We have cloned a full-length human ALDH1 cDNA and used retroviral vectors to transduce it into human (U937) and murine (L1210) hematopoietic cell lines that were then tested for resistance to maphosphamide, an active analogue of cyclophosphamide. Overexpression of the ALDH1 gene resulted in a significant increases in cyclophosphamide resistance in transduced L1210 and U937 cells (50% inhibition concentration [IC50], approximately 13 mumol/L). The resistant phenotype was specifically caused by ALDH1 overexpression as shown by its reversion by disulfiram, a specific ALDH1 inhibitor. ALDH1 transduction into peripheral blood human hematopoietic progenitor cells also led to significant increases (4- to 10-fold; IC50, approximately 3 to 4 mumol/L) in cyclophosphamide resistance in an in vitro colony-forming assay. These findings indicate that ALDH1 overexpression is sufficient to induce cyclophosphamide resistance in vitro and provide a basis for testing the efficacy of ALDH1 gene transduction to protect bone marrow cells from high-dose cyclophosphamide in vivo.

Cytoskeletons of central and peripheral neurons.

All eukaryotic cells have a cytoskeleton, consisting of microtubules, intermediate filaments and microfilaments. The cytoskeletal structure of cells and cell processes in the central nervous system is diverse. The generation of animal models in which specific mutations result in underexpression of overexpression of particular intermediate filament and microtubular proteins allows assessment of the possible role of cytoskeletal abnormalities in the neurodegenerative disorders. It is suggested that overexpression of filaments is likely to be the more significant process, but that neurofibrillary change, as recognized by the neuropathologist represents the final result of failure of any of a large number of molecular processes involved in cytoskeletal protein turnover.

BCL-6 and the molecular pathogenesis of B-cell lymphoma.

The results presented identify the first genetic lesion associated with DLCL, the most clinically relevant form of NHL. Although no proof yet exists of a role for these lesions in DLCL pathogenesis, the feature of the BCL-6 gene product, its specific pattern of expression in B cells, and the clustering of lesions disrupting its regulatory domain strongly suggest that deregulation of BCL-6 expression may contribute to DLCL development. A more precise definition of the role of BCL-6 in normal and neoplastic B-cell development is the goal of ongoing study of transgenic mice engineered either to express BCL-6 under heterologous promoters or lacking BCL-6 function due to targeted deletions. In addition to contributing to the understanding of DLCL pathogenesis, the identification of BCL-6 lesions may have relevant clinical implications. DLCL represent a heterogeneous group of neoplasms which are treated homogeneously despite the fact that only 50% of patients experience long-term disease-free survival (Schneider et al. 1990). The fact that BCL-6 rearrangements identify biologically and clinically distinct subsets of DLCL suggests that these lesions may be useful as markers in selection of differential therapeutic strategies based on different risk groups. Furthermore, the BCL-6 rearrangements can be used to identify and monitor the malignant clone with sensitive PCR-based techniques. Since clinical remission has been observed in a significant fraction of DLCL cases, these markers may serve as critical tools for sensitive monitoring of minimal residual disease and early diagnosis of relapse (Gribben et al. 1993).

Astrocyte process growth induction by actin breakdown.

cAMP analogues such as dibutyryl cAMP (dBcAMP) have been shown to induce the formation of processes in cultured primary astrocytes. We observe that the processes form by elongation as well as the previously reported retraction of cytoplasm around cytoskeletal elements. The most prominent cytoskeletal change that occurs in response to dBcAMP is a rearrangement of actin filaments characterized by a loss of cortical F-actin staining and the appearance of actin filament staining at the tips of the processes. If cortical actin filaments are disrupted with dihydrocytochalasin B, processes form that are similar to those induced by dBcAMP suggesting that the disruption of the cortical actin network is the pivotal step in process formation. Reorganization of the actin filament network in response to cAMP is accompanied by a decrease in phosphate incorporation into the regulatory light chain of myosin (MLC). Two selective inhibitors of MLC kinase (MLCK), ML-9 and KT5926, as well as a calmodulin antagonist (W7), which would also inhibit MLCK activation, all induce astrocytic process growth implicating MLCK as a control point in process initiation. We also found that dBcAMP and ML-9 both cause a decrease in the phosphate content of actin depolymerizing factor, suggesting that this protein and myosin light chain are the effectors of actin cytoskeleton reorganization and process growth.

High molecular weight tau: preferential localization in the peripheral nervous system.

Using epitope mapping we have demonstrated that a high molecular weight protein (Mr approximately 115 x 10(3)) present in brain and spinal cord is a member of the tau family of microtubule-associated proteins. Antibodies directed against the amino-terminal, middle and carboxyl-terminal portions of tau recognize this protein. A limited survey of neuronal tissues has shown that this high molecular weight tau protein is present in brain, spinal cord, dorsal root ganglia, dorsal and ventral roots and peripheral nerves. High molecular weight tau protein is expressed at higher levels in spinal cord than in brain and is the only form of tau detected in the adult peripheral nervous system.

Phosphorylation of the multidrug resistance associated glycoprotein.

Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.

Preparation and biological activity of taxol acetates.

Synthesis and biological activity of 2'-acetyltaxol and 7-acetyltaxol are reported. Activity is measured in vivo by cytotoxicity toward the macrophage-like cell line J774.2, and in vitro by promotion of microtubule assembly in the absence of exogenous GTP. Addition of an acetyl moiety at C-2' results in loss of in vitro activity but not cytotoxicity. The properties of 7-acetyltaxol are similar to those of taxol in its effects on cell replication and on in vitro microtubule polymerization. Therefore a free hydroxyl group at C-7 is not required for in vitro activity and this position is available for structural modifications.

Tubulin carbamoylation. Functional amino groups in microtubule assembly.

The characteristics of the carbamoylation of pig brain tubulin were examined by using the modification conditions with cyanate described previously [Mellado, Slebe + Maccioni (1980) Biochem. Int. I, 584--590]. The carbamoylation reaction resulted in an inhibition of microtubule assembly, which was dependent on the concentration of the modifying agent. This tubulin modification appears to inhibit the growth of microtubules. The presence of GTP did not protect tubulin against this inhibition. Electron microscopy showed a marked decrease in the number of tubules after carbamoylation, but no alterations were observed in the microtubule morphology. The incorporation of KN14CO into alpha- and beta-subunits with similar kinetics was also shown, and the carbamoylated residues were identified as epsilon-N-carbamoyl-lysine residues.

Isoacceptor glycine tRNA species during bovine myocardium development.

The isoacceptor patterns of glycyl-tRNAs from fetal bovine myocardium during development, and of adult cardiac muscle, have been studied by reverse-phase chromatography. 4 isospecies were detected and quantitative changes in their relative abundance were noted. Moreover, upon testing their efficiency in transferring glycine into polypeptides a differential utilization of the cognate tRNAs was observed.