Biomedical subjects

A Dritschilo

Publications and source records attributed to A Dritschilo.

At least 55 records · Page 3Linked to original sources

PubMed1996

Radiation-induced neoplastic transformation of human prostate epithelial cells.

We report the malignant transformation of human prostate epithelial cells (267B1) after multiple exposures to ionizing radiation. Carcinogenic progression of cells from immortal growth to anchorage-independent growth in soft agar to tumorigenicity in athymic mice resulted after a cumulative X-ray dose of 30 Gy. The tumors were characterized histologically as poorly differentiated adenocarcinomas, expressed prostate-specific antigen, and stained positive for keratin. No p53 or ras mutations were observed. Numerous chromosomal defects were noted on karyotypes after radiation exposure. However, chromosome 3 and 8 translocations were observed predominantly in the tumor outgrowths. These findings provide the first evidence of malignant transformation of human prostate epithelial cells exposed to ionizing radiation.

Animals↗

PubMed1996

An improved method of encapsulation of doxorubicin in liposomes: pharmacological, toxicological and therapeutic evaluation.

We describe here an improved method of encapsulating doxorubicin in liposomes using phosphatidylcholine, cholesterol and synthetic tetramyristoyl cardiolipin. With this new composition of lipids the entrapment of doxorubicin was found to be > 90%. Cytotoxicity studies using vincristine-resistant HL-60/VCR leukaemia cells showed that liposome-encapsulated doxorubicin reverses multidrug resistance 5-fold compared with conventional doxorubicin and at levels equivalent to that obtained using liposomes with natural cardiolipin. In normal mice, liposome-encapsulated doxorubicin was much less toxic than the conventional drug. A dose of 25 mg kg-1 i.v. of conventional doxorubicin produced 100% mortality in mice by day 14, whereas liposomal doxorubicin exhibited only 10% mortality by day 60. Liposomal doxorubicin demonstrated enhanced anti-tumour activity against murine ascitic L1210 leukaemia compared with conventional doxorubicin. At a dose of 15 mg kg-1, liposomal doxorubicin increased the median life span with 12 of 18 long-term (60 days) survivors compared with only 3 of 18 with conventional drug. Mice injected i.v. with liposomal doxorubicin had plasma levels 44-fold higher than conventional doxorubicin, producing significantly higher (P < 0.02) area under the plasma concentration curve. An altered tissue distribution was also observed with liposomal doxorubicin; cardiac tissue demonstrating at least 2-fold lower levels with liposomal doxorubicin probably accounting for its lower toxicity. This altered pharmacokinetics of liposome-encapsulated doxorubicin, providing enhanced therapeutic advantage and the ability to modulate multidrug resistance, could be useful in a clinical setting.

Animals↗

PubMed1996

Signal Transduction and Cellular Responses to Ionizing Radiation.

The current understanding of cellular responses to ionizing radiation fuses traditional radiobiological concepts with recent insight into underlying molecular mechanisms. Cell cycle arrest, induction of specific cascades of genes, and activation of DNA repair mechanisms have been reported after radiation exposure. Of particular importance is the mitogen-activated protein (MAP) kinase signaling pathway, in which the ras and raf oncogenes participate. Both of these genes have been implicated in the cellular resistance to killing by ionizing radiation. Furthermore, regulated NF-gammaB transcriptional activation may be important for the pleiotrophic responses of cells to ionizing radiation. Disruption of this signaling cascade may contribute to the radiation sensitivity syndrome seen in ataxia telangiectasia. In this review, these pathways are consolidated into ionizing radiation signal transduction pathways that lead from the cell membrane/cytosol to the nuclear DNA.

Journal Article↗

PubMed1996

Cytoskeletal changes during radiation-induced neoplastic transformation of human prostate epithelial cells.

We recently reported tumorigenic transformation of SV40-immortalized neonatal human prostate epithelial cells (267B1) by exposure to fractionated doses of X-rays. Altered morphology and anchorage independence were observed following two successive fractions of 2 Gy each (F3-SAC). Additional 2 Gy treatments to these non-tumorigenic cells to a total dose of 30 Gy resulted in radiation-transformed tumorigenic colonies (267B1-SXR). Malignant transformation of parental 267B1 cells was also achieved by consecutive 2 Gy exposures to a total dose of 30 Gy (267B1-XR). This study discusses the cytoskeletal changes in the F3-SAC, 267B1-XR and 267B1-SXR derivatives of these human prostate epithelial cells. Confocal and conventional fluorescence microscopy of filamentous actin showed numerous, well organized, evenly distributed stress fibers in the parental cells prior to irradiation, while the anchorage-independent cells and several tumorigenic derivatives exhibited poor stress fiber organization after radiation exposure. This disorganization of actin microfilaments in the radiation-transformed cells was also accompanied by changes in the expression of selective tropomyosin isoforms as judged by two-dimensional gel electrophoresis. These changes in actin organization and tropomyosin expression appear to be coincidental with morphological transformation and acquisition of tumorigenicity in the 267B1 cells following radiation exposure.

Actin Cytoskeleton↗

PubMed1996

Atomic force microscopy investigation of radiation-induced DNA double strand breaks.

We have used atomic force microscopy (AFM) to study radiation-induced DNA double strand breaks. Double-stranded plasmid DNA was irradiated with 18-MeV electrons in aqueous buffer, using a medical linear accelerator. Doses of 50, 100, 150, and 200 Gy were delivered to DNA samples, and atomic force microscopy was used to measure the length of each DNA fragment. From these measurements, we obtained the average length of the irradiated DNA for each sample and found a linear-quadratic relationship between the average length and radiation dose.

DNA↗

PubMed1995

Radiation-induced apoptosis of Ewing's sarcoma cells: DNA fragmentation and proteolysis of poly(ADP-ribose) polymerase.

Ewing's sarcoma (ES) cells express high levels of poly(ADP-ribose) polymerase (PADPRP) and are responsive to killing by ionizing radiation. We have determined that ionizing radiation induced a pronounced but reversible G2-M phase cell cycle arrest that was maximum by 24 h after exposure. Following the release from this block, floating cells began to appear. These floating cells were shown to be apoptotic by flow cytometric and DNA fragmentation analyses. We found that apoptosis is a significant component of radiation-induced death in ES cells and that this is accomplished in conjunction with proteolytic cleavage of PADPRP. Two fragments of M(r) 25,000 and M(r) 29,000 containing the PADPRP DNA-binding domain were identified in floating (apoptotic) cells, whereas only the full-length M(r) 116,000 native protein was detected in adherent cells that retained DNA intact. These data are consistent with PADPRP cleavage being an early step in the apoptotic cascade of biochemical events in ES cells after ionizing radiation exposure.

Apoptosis↗

PubMed1995

Isolation and characterization of SpTRK, a gene from Schizosaccharomyces pombe predicted to encode a K+ transporter protein.

A novel gene, SpTRK, has been isolated from DNA of the fission yeast Schizosaccharomyces pombe (Sp) by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a sequence fully conserved between the potassium transporter genes TRK1 and TRK2 of Saccharomyces cerevisiae (Sc). SpTRK is a single-copy gene located on Sp chromosome I. Nucleotide sequence analysis of the cloned gene identified an open reading frame (ORF) with coding capacity for a protein of 833 amino acids (aa). The predicted SpTRK aa sequence showed a high level of conservation relative to the potassium transporters of Sc and Saccharomyces uvarum (Su), particularly within their transmembrane (TM) domains and in aa required for their ion transport functions. A single SpTRK transcript of about 2.7 kb is expressed at high levels in exponentially growing Sp cells, but it is downregulated in cells from stationary cultures.

Amino Acid Sequence↗

PubMed1995

Enhanced expression of calreticulin in the nucleus of radioresistant squamous carcinoma cells in response to ionizing radiation.

Ionizing radiation has been shown to modulate gene and protein expression as well as cellular signal transduction pathways. However, the biochemical and molecular mechanisms that underlie the cellular response to radiation are not fully understood. The effects of ionizing radiation on the expression of nuclear proteins have now been investigated in radiorestistant human head and neck squamous carcinoma cells (SQ-20B cells) using the techniques of two-dimensional PAGE, silver staining, and computer-assisted quantitative analysis. Radiation (600 cGy) induced the expression of 10 proteins and suppressed the expression of 5 proteins in the nuclei of SQ-20B cells as detected 4 h after treatment. Electroelution and NH2-terminal amino acid sequence analysis revealed that one of the radiation-induced proteins was the Ca(2+)-binding protein calreticulin. The expression of calreticulin was increased approximately 6-fold in the nuclei of irradiated SQ-20B cells. Calreticulin and the other proteins whose expression was affected by radiation may contribute to the radioresistance phenotype of SQ-20B cells.

Amino Acid Sequence↗

PubMed1995

Correction of radiation sensitivity in ataxia telangiectasia cells by a truncated I kappa B-alpha.

Cells from patients with ataxia telangiectasia (AT) are hypersensitive to ionizing radiation and are defective in the regulation of DNA synthesis. A complementary DNA that corrects the radiation sensitivity and DNA synthesis defects in fibroblasts from an AT group D patient was isolated by expression cloning and shown to encode a truncated form of I kappa B-alpha, an inhibitor of the nuclear factor kappa B (NF-kappa B) transcriptional activator. The parental AT fibroblasts expressed large amounts of the I kappa B-alpha transcript and showed constitutive activation of NF-kappa B. The AT fibroblasts transfected with the truncated I kappa B-alpha expressed normal amounts of the I kappa B-alpha transcript and showed regulated activation of NF-kappa B. These results suggest that aberrant regulation of NF-kappa B and I kappa B-alpha contribute to the cellular defect in AT.

Amino Acid Sequence↗

PubMed1995

Co-regulated expression of dbl and poly(ADP-ribose) polymerase in Ewing's sarcoma cells and dbl-transformed NIH3T3 fibroblasts.

Poly(ADP-ribose) polymerase (PADPRP) is a ubiquitous enzyme constitutively expressed at low levels in the majority of eukaryotic cells, including most mammalian tumors and tumor-derived cell lines. Overexpression of PADPRP following the introduction of cDNA recombinant constructs into various cell types results in cell death. An exception to this effect are Ewing's sarcoma (ES) cells, which have been shown to contain elevated steady-state levels of PADPRP mRNA and high constitutive levels of protein and polymerase activity. In fact, this excess of PADPRP has been suggested to participate in the intrinsic radiosensitivity of Ewing's sarcomas, a highly malignant childhood bone tumor frequently curable with radiotherapy. It appears that ES cells might possess a hitherto unknown mechanism(s) by which PADPRP overexpression is controlled and made compatible with cell survival and proliferation. In order to investigate the contribution of other genetic alterations to PADPRP regulation in ES cells, we analysed the expression levels of PADPRP and of other genes, such as oncogenes and tumor suppressor genes, which may enhance the proliferative potential of ES cells. We have detected a positive correlation between the expression levels of the DNA-repair enzyme poly(ADP-ribose) polymerase and the dbl proto-oncogene in Ewing's sarcoma cells. The co-regulated expression of these genes has been established in NIH3T3 cells transformed by the human dbl oncogene or by overexpression of the dbl proto-oncogene. In both instances, the increase in dbl expression resulted in elevated levels of PADPRP mRNA and polymerase activity. The dbl oncogene was more efficient than the proto-oncogene in upregulating PADPRP expression. The inability of other oncogenes to upregulate PADPRP upon transformation of NIH3T3 cells demonstrated the specificity of the dbl in the process. Transfection of dbl-transformed NIH3T3 cells with retroviral PADPRP vectors resulted in the establishment of clones with PADPRP levels higher than those detectable in untransformed NIH3T3 cells transfected with the same retroviral constructs. These results suggest that dbl plays a role in the mechanism by which mammalian cells autoregulate their endogenous levels of PADPRP. Post-translational modification of the dbl or proto-dbl proteins by cytoplasmic PADPRP does not participate in the mechanism(s) underlying the observed PADPRP/dbl co-regulation.

3T3 Cells↗

PubMed1995

Modification of nucleolar protein B23 after exposure to ionizing radiation.

The responses of cells to ionizing radiation include induction and/or suppression of the expression of genes and proteins. In our investigations of alterations in cellular protein expression in response to ionizing radiation, we have used the techniques of two-dimensional polyacrylamide gel electrophoresis and silver staining. We compared the nuclear protein profiles of control and irradiated (6 Gy, 4 h postirradiation) radioresistant squamous carcinoma cells (SQ-20B) and observed an alteration in the expression of a 40 kDa protein: control nuclei express a protein isoform with pI values between 5.4-5.8, while irradiated nuclei express a more acidic variant with pI values between 5.2-5.5. Using the cyanogen bromide/O-phthalaldehyde method followed by microsequencing analysis, we obtained an internal amino acid sequence and identified the 40 kDa protein as nucleolar protein B23. Western blotting experiments confirmed the internal amino acid sequencing results and showed both species (control, 5.4-5.8, irradiated, 5.2-5.5) to be recognized by an anti-B23 monoclonal antibody. Radiolabeling of control and irradiated samples with [32P]NAD or [32P]orthophosphoric acid revealed the acidic species of B23 to be both ADP-ribosylated and phosphorylated. Therefore, exposure of SQ-20B cells to radiation results in the increase in expression of an ADP-ribosylated and phosphorylated species of B23.

Adenosine Diphosphate Ribose↗

PubMed1994

Expression of thymidine kinase is essential to low dose radiation resistance of rat glioma cells.

We have found that thymidine kinase expression is a major radioresponse determinant in rat glioma cells. Cells that lack thymidine kinase expression are significantly more radiosensitive relative to the wild-type cells. The degree of sensitization is large, particularly at the dose levels used in fractionated radiotherapy. The difference in low dose survival can be accounted for by a marked difference in the ability of the cells to undergo repair of sublethal damage. When herpes thymidine kinase was introduced into the thymidine kinase-deficient mutant cells, radioresistance was partially restored, and sublethal damage repair was also enhanced. All other radiobiological responses, including DNA double-strand break repair, potentially lethal damage repair, G2 arrest, and cell cycle distribution, appeared similar among the cell lines. These data suggest that the thymidine kinase enzyme or its cellular gene may be an excellent therapeutic target to increase radiosensitivity and thereby, to enhance the radiocurability of malignant brain gliomas.

Animals↗

PubMed1994

ADP-ribosylation of heterogeneous ribonucleoproteins in HeLa cells.

Poly and mono (ADP-ribosyl)transferases catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD) to acceptor proteins. Protein substrates of ADP-ribosylation reactions were investigated in human cervical carcinoma (HeLa) cells in the exponential phase of growth. Permeabilized cells were incubated with [32P]-NAD and ADP-ribosylated proteins were detected by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and autoradiography. Microsequencing of two coomassie blue-stained proteins corresponding to 32P-labeled proteins on autoradiographs revealed sequence identity with heterogeneous ribonucleoproteins (hnRNPs) A1 and A2/B1, consistent with the isoelectric points, molecular sizes, and 2D-PAGE map locations of these proteins. ADP-ribosylation of hnRNPs may thus serve to modulate the activity of these proteins in the nucleus.

Adenosine Diphosphate Ribose↗

PubMed1994

Fibroblast growth factor-4 enhanced G2 arrest and cell survival following ionizing radiation.

Fibroblast growth factors (FGFs) bind to cell membrane receptors and activate signal transduction pathways related to cell growth, angiogenesis, and tumorigenesis. FGFs have been shown to be abundantly expressed in some of the human tumors, which are known to be poorly responsive to radiation therapy. Using adrenal cortical carcinoma cells genetically engineered to express FGF-4, we have tested cellular survival following exposure to ionizing radiation. We report here that FGF-4 enhances cellular capacity to survive ionizing radiation. Furthermore, cell cycle analysis shows a pronounced increase in the duration of G2 arrest, suggesting perturbation of a cell cycle checkpoint. These findings implicate fibroblast growth factor-mediated signal transduction in cellular resistance of human tumors to radiation therapy.

Cell Survival↗

PubMed1994

Elongation factor 1 delta is enhanced following exposure to ionizing radiation.

We have used the technique of differential display of mRNA to investigate gene expression patterns in a human squamous carcinoma cell line, SCC-35, following exposure to ionizing radiation. We observed that elongation factor 1 delta (EF-1 delta) expression increased within 2 h of radiation exposure with maximum expression at 8 h. These data identify EF-1 delta as a radiation-inducible gene. Cell cycle analysis shows that EF-1 delta expression enhancement precedes G2 arrest in these cells, suggesting that EF-1 delta may participate in the G2-M cell cycle checkpoint.

Base Sequence↗

PubMed1994

Functional expression of human poly(ADP-ribose) polymerase in Schizosaccharomyces pombe results in mitotic delay at G1, increased mutation rate, and sensitization to radiation.

The activity of poly(ADP-ribose) polymerase (PADPRP), a chromatin-associated enzyme present in most eukaryotic cells, is stimulated by DNA strand breaks, suggesting a role for the enzyme in the cellular response to DNA damage. However, the primary function of PADPRP remains unknown. We have selected Schizosaccharomyces pombe as a simple eukaryotic system in which to study PADPRP function because this fission yeast shares with mammalian cells important cellular features possibly associated with poly-(ADP-ribos)ylation pathways. We investigated the existence of an endogenous yeast PADPRP by DNA and RNA hybridization to mammalian probes under low-stringency conditions and by PADPRP activity assays. Our data indicate that fission yeasts are naturally devoid of PADPRP. We therefore isolated S. pombe strains expressing PADPRP by transformation with a human full-length PADPRP cDNA under the control of the SV40 early promoter. The human PADPRP construct was transcribed and translated in S. pombe, generating a major transcript of the same size (3.7 kb) as that detected in mammalian cells and a 113-kDa polypeptide, identical in size to the native human PADPRP protein. Yeast recombinant PADPRP was enzymatically active and was recognized by antibodies to human PADPRP. S. pombe cells expressing PADPRP (SPT strains) showed a stable phenotype that was characterized by: (i) cell cycle retardation as a result of a specific delay at the G1 phase, (ii) decreased cell viability in stationary cultures, (iii) enhanced rates of spontaneous and radiation-induced ade6-ade7 mutations, and (iv) increased sensitivity to radiation. SPT strains may prove efficient tools with which to investigate PADPRP functions in eukaryotic cells.

Dose-Response Relationship, Radiation↗

PubMed1994

A new procedure for the preparation of liposomal doxorubicin: biological activity in multidrug-resistant tumor cells.

We describe a new procedure for the preparation of liposomal doxorubicin. Doxorubicin can be efficiently complexed to preformed or lyophilized cardiolipin-containing liposomes. Complex formation was performed by vigorous vortexing. As much as 96.8% of the initial drug quantity may be bound to those liposomes under optimal incubation conditions (4 h at 37 degrees C). The binding study showed the presence of two levels of specific binding (dissociation constants, 28 +/- 8 microM and 1.0 +/- 0.3 mM). The drug is firmly integrated in the liposome-membrane lipid bilayer rather than binding at the surface. Cytotoxicity studies using tumor cells revealed efficient drug delivery using liposome-complexed doxorubicin. This new liposomal doxorubicin preparation reverses multidrug resistance in MCF-7/ADR and CH LZ cells at levels equivalent to that obtained with a previously described liposome-encapsulated doxorubicin preparation, showing that the drug is integrated as well in the liposome carrier and is transported as well into cells. Increased concentration of liposomes at the subcytotoxic level in liposome-complexed doxorubicin enhances drug cytotoxicity in multidrug-resistant CH LZ cells as compared with liposome-encapsulated drug. This new preparation for liposomal doxorubicin may be carried out immediately prior to clinical administration, offering advantages in terms of cost and stability.

Breast Neoplasms↗

PubMed1994

Transformation of human epidermal keratinocytes with fission neutrons.

The biological effects of exposures to high LET radiations have particular relevance to radiation protection and risk assessment. Since most cancers are of epithelial origin, it is important to obtain a better understanding of radiation-induced oncogenic transformation in this cell type. Accordingly we have initiated studies to determine whether immortalized human epidermal keratinocytes (RHEK) can be transformed with high LET radiations. Exponentially growing RHEK cells were treated with single doses (1, 10, 25, 50 and 100 cGy) of 0.85 MeV fission neutrons from the Janus reactor. Neutron exposure led to the development of morphologically altered cells and foci formation after 6 weeks at confluence. These transformed cultures grew with an increased saturation density, exhibited anchorage-independent growth and formed tumors in athymic mice. Single-strand conformational polymorphism analysis and DNA sequencing demonstrated the absence of point mutations in codons 12/13 and 61 in the Ha-ras, Ki-ras, or N-ras genes and exons 4-9 of the p53 tumor suppressor gene. These studies demonstrate that high LET radiations (fission neutrons) can transform immortalized human epithelial cells to a malignant phenotype that does not appear to involve mutations in either the cellular p53 or ras genes.

Animals↗