Biomedical subjects

A Dritschilo

Publications and source records attributed to A Dritschilo.

At least 91 records · Page 5Linked to original sources

PubMed1992

Reliable and efficient direct sequencing of PCR-amplified double-stranded genomic DNA template.

Modified PCR amplification and direct sequencing procedures for the double-stranded genomic DNA template are described. Advantages of the approach we describe are: background artifact bands previously observed using high-molecular-weight DNA as a template were eliminated by this protocol; no gel purification or subcloning of the PCR-amplified double-stranded fragment was required prior to direct sequencing; and sequences of 300 nucleotides can be easily read even after a single loading. The successful use of the modified dideoxynucleotide chain-termination method for direct sequencing of both strands demonstrates the efficiency of this technique for removing sequencing artifacts and for producing reliable sequence data.

Base Sequence↗

PubMed1991

Biological consequences of gene regulation after ionizing radiation exposure.

Ionizing radiation is a ubiquitous environmental mutagen and carcinogen widely used in cancer therapy. However, little is known about the induction of cellular signaling events and specific gene expression after radiation exposure. We review the accumulating evidence that ionizing radiation induces signal transduction pathways involving activation of protein kinase C and a program of genetic events that may contribute to the biological effects of x rays.

Base Sequence↗

PubMed1991

Baseline sister chromatid exchange in human cell lines with different levels of poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase is a chromatin enzyme which adds long chains of ADP-ribose to various acceptor proteins in response to DNA strand breaks. Its primary function is unknown; however, a role in DNA repair and radiation resistance has been postulated based largely on experiments with enzyme inhibitors. Recent reports of mutant cell lines, deficient in poly(ADP-ribose) polymerase activity, have supported previous studies with inhibitors, which suggests the involvement of poly(ADP-ribose) polymerase in maintaining baseline levels of sister chromatid exchanges. Mutant cells with even slightly depressed enzyme levels show large elevation of baseline sister chromatid exchanges. Since intracellular poly(ADP-ribose) polymerase levels can vary greatly between different nonmutant cell lines, we surveyed levels of baseline sister chromatid exchange in normal and tumor human cell lines and compared them with endogenous levels of poly(ADP-ribose) polymerase. Despite 10-fold differences in poly(ADP-ribose) polymerase, the baseline level of sister chromatid exchanges remained relatively constant in the different cell lines (0.13 +/- 0.03 SCE/chromosome), with no indication of a protective effect for cells with high levels of the enzyme.

Humans↗

PubMed1990

Evidence for the multistep nature of in vitro human epithelial cell carcinogenesis.

In keeping with the multistep development of human cancer in vivo, a stepwise approach to neoplastic transformation in vitro presents a reasonable strategy. We have recently developed an in vitro multistep model suitable for the study of human epithelial cell carcinogenesis. Upon infection with the adenovirus 12-simian virus 40 hybrid virus, primary human epidermal keratinocytes acquired an indefinite life span in culture but did not undergo malignant conversion. Subsequent addition of Kirsten murine sarcoma virus and human ras oncogene or chemical carcinogens (N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline 1-oxide) to these cells induced morphological alterations and the acquisition of neoplastic properties. Subsequently it was found that this line could be transformed neoplastically by a variety of retrovirus-containing H-ras, bas, fes, fms, erbB, and src oncogenes. In addition, we found that the immortalized human epidermal keratinocyte (RHEK-1) line can be transformed neoplastically by exposure to ionizing radiation. Thus, this in vitro system may be useful in studying the interaction of a variety of carcinogenic agents and human epithelial cells. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of viruses, oncogenes, chemical carcinogens, or X-ray irradiation and support a multistep process for neoplastic conversion.

Adenoviridae↗

PubMed1990

Enhanced poly(adenosine diphosphate ribose) polymerase activity and gene expression in Ewing's sarcoma cells.

Ewing's sarcoma (ES) is a highly malignant childhood bone tumor and is considered curable by moderate doses of radiotherapy. The addition of chemical inhibitors of the activity of the nuclear enzyme poly(adenosine diphosphate ribose) [poly(ADPR)] polymerase to ES cells in culture results in increased cell killing, a phenomenon called "inhibitor sensitization." Since poly(ADPR) polymerase is thought to be associated with DNA repair, it has been suggested that ES cells and other inhibitor-sensitized cells may have a reduced capacity for polymer synthesis resulting in deficient postirradiation recovery. We present here the unexpected observation that in comparison to other cell lines tested, ES cells exhibit a high enzyme activity, higher constitutive levels of the protein, and elevated levels of its mRNA transcript for poly(ADPR) polymerase. No gross amplifications or rearrangements of the gene were observed; however, regulation of poly(ADPR) polymerase in these tumor cells takes place at the level of the gene transcript.

Blotting, Northern↗

PubMed1990

A randomized study of the efficacy of adjuvant local graft irradiation following renal transplantation.

A prospective randomized study investigating the effectiveness of adjuvant local graft irradiation (LGI) following renal transplantation was performed at Georgetown University Hospital from 1983 until 1988. One hundred and thirty-eight patients were enrolled in the study with 117 patients receiving cadaver kidney transplantations and 21 patients receiving living related kidney transplantations. Seventy-one patients were randomized to receive adjuvant local graft irradiation consisting of 600 cGy in four fractions with chemical immunosuppression whereas the remaining 67 patients received chemical immunosuppression only (control group). The two groups were comparable at entry with respect to potentially important prognostic variables. Median follow-up for all patients was 30 months. The 3-year actuarial allograft success rate was 75% and 68% for the local graft irradiation and control groups, respectively. A nonsignificant trend favoring the irradiated group was noted. Subgroup analysis of the 21 recipients of kidneys from living related donors suggested an improvement in allograft survival for the local graft irradiation arm. Cadaver allograft survival was not significantly different between the two treatment arms. There was no apparent benefit in kidney function or time to the first rejection episode in the group receiving local graft irradiation.

Adult↗

PubMed1990

Neoplastic transformation of immortalized human epidermal keratinocytes by ionizing radiation.

Efforts to investigate the progression of events that cause human cells to become neoplastic in response to ionizing radiation have been aided by the development of tissue culture systems of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus type 12 and simian virus 40 have been transformed by exposure to x-ray irradiation. Such transformants showed morphological alterations, formed colonies in soft agar, and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes exposed to radiation in this manner failed to show any evidence of transformation. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of a DNA tumor virus and radiation, indicating a multistep process for radiation-induced neoplastic conversion. This in vitro system may be useful as a tool for dissecting the process of radiation-induced neoplastic transformation of human epithelial cells and for detecting previously unreported human oncogenes.

Animals↗

PubMed1990

Constraints to DNA unwinding near radiation-induced strand breaks in Ewing's sarcoma cells.

Ewing's sarcoma cell lines were compared to other cell lines for induction of DNA strand breaks by ionizing radiation and their ability to repair those breaks. The alkali-unwinding assay and alkaline sucrose gradient analysis were used for these studies. The alkali-unwinding assay revealed that the amount of DNA unwound per strand break in Ewing's sarcoma cells was less than for other cells and was not influenced by high-salt denaturation conditions. Ewing's sarcoma cells had similar induction and repair rates for strand breaks compared with other cell lines. The kinetics of unwinding suggests there are constraints to DNA unwinding in the chromatin of Ewing's sarcoma cells, possibly related to high levels of poly(ADP-ribose) polymerase in these cells.

Cell Line↗

PubMed1989

Poly(ADP-ribose)-mediated post-translational modification of chromatin-associated human topoisomerase I. Inhibitory effects on catalytic activity.

We have investigated the association of human topoisomerase I with poly(ADP-ribosylated) domains of chromatin and the effects of this modification on the enzyme activity. In vitro poly(ADP-ribosylation) assays demonstrated that this enzyme was one of the major acceptors for this chromatin-dependent post-translational modification. Western blotting procedures using antibody to topoisomerase I indicated that under extensive poly(ADP-ribosylation) conditions, where a majority of poly(ADP-ribose) acceptor molecules form aggregates, the major population of the topoisomerase I associated with chromatin was apparently non-aggregated. The catalytic activity of the topoisomerase I associated with the poly(ADP-ribosylated) chromatin was 3-5-fold inhibited. Additionally, antibody to poly(ADP-ribose) was used to immunofractionate selectively the modified domains of chromatin. Our data suggests the presence of topoisomerase I, both adjacent and distal to the poly(ADP-ribosylated) sites of chromatin. Unmodified and a significant portion of the modified species of enzyme migrated as approximately 100-kDa proteins. However, the modified form of topoisomerase was noted to be catalytically less active as compared to the enzyme bound to the non-poly(ADP-ribosylated) nucleosomes. These results provide evidence, at the cellular level, for the poly(ADP-ribosylation)-mediated regulation of human topoisomerase I and suggest a functional significance for poly(ADP-ribosylation) in topoisomerase-related processes (replication, transcription, and recombination) in eukaryotes.

Blotting, Western↗

PubMed1989

Sensitivities of NIH/3T3-derived clonal cell lines to ionizing radiation: significance for gene transfer studies.

Rodent cells are frequently used as recipients in experiments involving gene transfer, isolation, and characterization. The present studies were designed to investigate the clonal responses to ionizing radiation of NIH/3T3 cells subjected to DNA-mediated gene transfer. Radiation sensitivity (D0) values were determined for the parental NIH/3T3 cell strain, six clonal cell lines transfected with DNA from radiation-resistant human tumor cells, and six nontransfected clonal cell lines. The radiation sensitivities of four transfected and two nontransfected clonal cell lines differed significantly from parental NIH/3T3 cells (P less than 0.05). Detailed karyotype analysis of two nontransfected clonal cell lines with differing radiation sensitivities showed variation in chromosomal composition. Specifically, a minute chromosome was observed to segregate consistently (in 49 of 50 metaphases) with the genome of one NIH/3T3 clone (D0 2.07 Gy) and was completely absent (from 50 metaphases) in another NIH/3T3 clone (D0 1.06 Gy). In the parental NIH/3T3 strain (D0 2.02 Gy) 10% of cells (3 of 30 metaphases) had such minute chromosomes. These findings demonstrate that the clonal cellular heterogeneity of NIH/3T3 cells is characterized by genotypic and phenotypic variations which must be considered in the experimental design involving gene transfer and expression.

Animals↗

PubMed1989

Effect of antisense c-raf-1 on tumorigenicity and radiation sensitivity of a human squamous carcinoma.

Antisense RNA-mediated inhibition of gene expression was used to investigate the biological function of the c-raf-1 gene in a radiation-resistant human squamous carcinoma cell line, SQ-20B. S1 nuclease protection assays revealed that transfection of full-length raf complementary DNA in the antisense orientation (AS) leads to a specific reduction (greater than tenfold) of steady-state levels of the endogenous c-raf-1 sense (S) transcript in SQ-20B cells. In nude mice, the malignant potential of SQ-20B cells transfected with raf (S) was significantly increased relative to that of SQ-20B cells transfected with raf (AS). SQ-20B cells containing transfected raf (S) maintained a radiation-resistant phenotype as compared to those cells harboring the AS version, which appeared to have enhanced radiation sensitivity. These data indicate that the reduced expression of endogenous c-raf-1 is sufficient to modulate the tumorigenicity and the radiation-resistant phenotype of SQ-20B cells, thus implicating c-raf-1 in a pathway important to the genesis of this type of cancer.

Animals↗

PubMed1989

A dot-blot method for screening polyclonal and monoclonal antisera to poly(ADP-ribose).

ADP-ribosylation reactions play a key role at several points in cellular regulation and repair of DNA damage. The use of polyclonal or monoclonal antisera to poly(ADP-ribose) as probes to localize the site(s) of action of the polymer offers a promising tool for these studies. We report here a simple, sensitive method for detection and titration of these antisera to poly(ADP-ribose) using nitrocellulose membrane (NC) as a support for a dot-blot analysis. We take advantage of the fact that a highly labeled poly(ADP-ribose) preparation can be obtained by incubation of a 0.3 M KCl extract prepared from calf thymus nuclei with 32P-NAD. Such a preparation of labeled antigen is used as a reagent to detect the positive antibody spots on the NC with negligible background. Subsequent titration of the antisera and their semi-quantitative evaluation are also feasible using the dot-blot method. The sensitivity of the assay is only limited by the specific activity that can be achieved for the labeled polymer prepared as the antigen probe. The advantage of this method is that it eliminates the need to prepare pure, highly radiolabeled polymer as well as the fact that several samples can be handled on the membrane simultaneously. We demonstrate application of this technique for screening sera from patients with systemic lupus erythematosus (SLE) for anti-poly(ADP-ribose) antibodies. Further, we also extend the use of these sera for immunoquantitation of ADP-ribosylated proteins in six human tumor cells in tissue culture.

Animals↗

PubMed1989

Carcinogenic effects of scattered dose associated with radiation therapy.

Ionizing radiation has long been recognized as a weak carcinogen, and the risk of developing a radiation induced neoplasm after exposure to therapeutic radiation has been established. In the case of therapeutic radiation for treatment of existing malignancies, concomitant risk factors for second malignancies can confound the effect of radiation alone. This study presents a model for evaluating the isolated contribution of ionizing radiation to the induction of second malignancies in cancer patients, and presents estimates of the expected number of second malignancies induced in selected sensitive sites by scatter radiation during radiotherapy for cancer. The study focused on the year 1987, during which it was estimated that 192,761 new cancer patients received radiotherapy as part of their initial treatment plan. The model predicted that radiation may induce 63-84 secondary breast cancers, 64-72 secondary thyroid cancers, 94-157 secondary lung cancers, and 489-707 secondary leukemias over the remaining lifetime of this patient population. This represents a lifetime incidence of 0.7% for leukemia, and 0.3% for the solid tumors. This incidence must be placed in perspective with the current concepts of cancer management, such as combined modality therapy that may carry a risk of carcinogenesis greater than either modality alone, and when the alternatives to radiotherapy may be nonexistent or may be cosmetically or functionally undesirable. The information presented may be used in weighing the risks and benefits of alternative treatments for cancer.

Female↗

PubMed1989

Modulation of multidrug resistance in Chinese hamster cells by liposome-encapsulated doxorubicin.

A Chinese hamster cell line (LZ), selected for multidrug resistance (MDR), exhibits a 3,000-fold resistance to doxorubicin, compared to parental V-79 cells. These drug resistant cells have amplified MDR genes, overexpress P-glycoprotein, and in the presence of doxorubicin show reduced intracellular drug accumulation. Using liposome-encapsulated doxorubicin (Rahman et al. Cancer Res. 45:796-803; 1985), we observed partial reversal of the resistance of LZ cells to this drug and a higher intracellular drug accumulation, compared to free drug. Parental V-79 cells, however, did not exhibit differences in survival or in drug accumulation when treated with encapsulated or free doxorubicin. Comparison of the effect of liposome-encapsulated doxorubicin with that of verapamil in reversing drug resistance showed that the liposomal preparation was as effective as verapamil used at its maximum clinically relevant concentration (1.5 microM). These results suggest that the use of liposomes as carriers of anticancer drugs may offer a strategy for overcoming MDR in tumor cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Quantitation of strand breaks in human DNA using 32P-Alu hybridization: application to exponential and plateau-phase cells.

A sensitive quantitation of DNA (0.2 to 10 ng) can be achieved using a 32P-labeled Alu probe to hybridize human DNA spotted onto nylon membrane. This allows the determination of radiation-induced single-strand breaks without the use of [3H]thymidine prelabeling of cells in culture. The sensitivity of this technique in HeLa cells is comparable to results obtained using the alkaline unwinding technique. The method is applicable to cells in both exponential and plateau phases of growth.

Cesium Radioisotopes↗

PubMed1988

Heterogeneity of radiation response of a parent human epidermoid carcinoma cell line and four clones.

We examined the radiobiological parameters of a parent tumor cell line and four tumor clones of a human skin squamous cell carcinoma. The parent line and all clones have a tumor morphology, aneuploid karyotype, and the ability to passage infinitely in vitro. The parent cell line and three of four clones formed tumors in nude mice. The parent line, SCC-12, has a D0 of 1.59 Gy and an n of 7.5. In the four clones, D0 ranges from 1.31 Gy to 2.66 Gy and n ranges from 2.1 to 22.8. Potentially lethal damage repair (PLDR) following 11 Gy ranges from 1.7 to 13.1, at 24 hours, however PLDR following equitoxic doses of radiation is similar. The mean inactivation dose value (D) in the parent tumor cell line is 3.48 and ranges from 3.31 to 4.17 in the tumor clones. Radiobiological interpretation of heterogeneity may complicate predictive assays for clinical radiotherapy.

Carcinoma, Squamous Cell↗

PubMed1988

Intraoperative radiation therapy of hepatic metastases: technical aspects and report of a pilot study.

Surgical resection of hepatic metastases offers long-term survival, and possible cure, for selected patients with colorectal carcinoma. Fifty percent of patients considered candidates for resection are found to have disease confined to the liver. The resections necessary are often more extensive than predicted preoperatively, which provides an opportunity for innovative approaches using radiation therapy. The intraoperative radiation therapy technique presented here offers the ability to control multiple metastatic deposits in patients not deemed resectable. This is achieved using remote afterloading interstitial (Ir-192) radiation therapy to deliver tumoricidal radiation doses to limited volumes within the liver. The technique was used to treat 11 patients in a pilot study, delivering radiation doses of 20 Gy to the periphery of predetermined target volumes in a single treatment. The number of metastatic deposits treated ranged from 2 to 11 separate tumors with maximum diameters from 3 to 9 cm (median 6 cm). Hospitalizations were from 6 to 23 days (median 8) with only one patient experiencing a surgically related complication (wound dehiscence and pneumonia). There were no radiation related complications on follow-up to 18 months. Biopsies of two treated sites in a patient undergoing reoperation confirmed control of tumors by this procedure. This technique is offered as a standby procedure to patients undergoing exploration for hepatic resection at our institution.

Aged↗

PubMed1988

In vitro radiobiological parameters of human sarcoma cell lines.

In vitro radiobiologic survival parameters have been determined for 7 human osteosarcoma, 5 human soft tissue and bone sarcomas, and 4 Ewing's sarcoma cell lines. The mean D0 values were 99.5 +/- 11.6 cGy, 90.5 +/- 7.7 cGy and 95.8 +/- 7.9 cGy for osteosarcomas, soft tissue and bone sarcomas and Ewing's sarcomas, respectively. These in vitro survival data do not predict the clinical radiation resistance generally attributed to osteosarcomas and soft tissue and bone sarcomas, and do not differ substantially from the results obtained with the clinically radioresponsive Ewing's sarcomas.

Bone Neoplasms↗