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Biomedical subjects

M Dietel

Publications and source records attributed to M Dietel.

At least 145 records · Page 8Linked to original sources

[Polymerase chain reaction in diagnostic pathology].

DNA is a very stable molecule and fixation as well as routine histological processing does not destroy its molecular structure. Hence the possibility exists to use this material for molecular genetic analyses. The polymerase chain reaction (PCR) opens the chance to amplify DNA to huge amounts from pathological paraffin and plastic blocks and to use this DNA for further examinations, such as detection of mutations or translocations in malignant tumors, e.g. t(14;18) in follicular lymphomas, bcr/abl in chronic myeloic leukemia, t(11;22) in Ewing sarcomas or of the ras-gene in colon cancer, overexpression of tumor related mRNA, e.g. mdr1-mRNA of the multidrug associated P-Glycoprotein, or detection of foreign DNA from viruses or bacteriae, as well as analysis of hereditary diseases. PCR in its various forms (conventional PCR, PCR with direct sequencing, reverse transcriptase (RT)-PCR, nested primer PCR, quantitative RT-PCR, inverse PCR, degenerated primer (DOP) PCR, in situ PCR, in situ RT-PCR etc.) has proven to supplement routine diagnostic work in many occasions, however, it has to be emphasized that up to now there exists no example for a complete replacement of histological or immunhistological examination by PCR. As consequence, histology remains the first and most important step towards a relevant diagnosis supplemented e.g. by PCR and other techniques of molecular biology.

Communicable Diseases↗

Reversal of multidrug resistance in Friend leukemia cells by dexniguldipine-HCl.

Dexniguldipine-HCl (DNIG)--a prospective clinical modulator of p170-glycoprotein (pgp170)-mediated multidrug resistance (MDR)--was evaluated in a drug-accumulation assay in MDR murine leukemia cell strain F4-6RADR expressing pgp170. The compound elevated low accumulation of either doxorubicin (DOX), daunorubicin (DNR), or mitoxantrone (MITO) in resistant F4-6RADR cells to the very levels observed in drug-sensitive F4-6 precursor cells. In parallel with the increase in DNR content (F4-6RADR, solvent: 303 +/- 27 pmol/mg protein; DNIG (3.3 mumol/l): 1,067 +/- 174 pmol/mg protein; F4-6P, solvent: 948 +/- 110 pmol/mg protein; n = 8-9, SEM), the amount of DNR tightly bound to the acid precipitate pellet obtained from F4-6RADR (i.e., protein, DNA, RNA) increased 3.9-times to the levels observed in sensitive F4-6 cells. The main pyridine metabolite of DNIG displayed similar activity. Concentration-response analysis revealed that DNIG and R,S-verapamil (VER) induced 100% reversal of the DNR accumulation shortage associated with the MDR phenotype but DNIG was 8 times more potent than VER (50% inhibitory concentration (IC50), 0.73 vs 5.4 mumol/l). In keeping with the accumulation assay, DNIG was about 10 times more potent than VER in sensitizing F4-6RADR cells to the cytostatic and cytotoxic effects of DNR in proliferation assays. In conclusion, DNIG is a potent in vitro modulator, improving (a) the accumulation of anthracycline-like cytostatics, (b) drug access to cellular binding sites, and (c) the cytostatic action of DNR in F4-6RADR leukemia cells of the MDR phenotype.

Animals↗

In vitro prediction of cytostatic drug resistance in primary cell cultures of solid malignant tumours.

The in vitro monolayer proliferation assay (MP-assay) described here enables predictive determination of the efficacy of anticancer drugs considered for clinical application. The assay was designed (1) to achieve a high plating efficiency, (2) to adapt in vitro growth as close as possible to in vivo conditions, and (3) to prove that the cells in vitro correspond with the in vivo tumour cells they were derived from. From 452 freshly explanted or biopsied tumours, 321 (71%) proliferating cultures could be established. To prove malignant origin of the incubated cells each strain was characterised by DNA-cytophotometry for aneuploidy and by immunocytochemistry for marker proteins. Drug potency was determined by comparing the number of living cells in drug-treated cultures with non-treated controls. Drug concentrations in vitro corresponded with those achievable in tumour tissue and thus represented clinically relevant levels. Growth inhibition in vitro was correlated with in vivo tumour response. Two hundred in vitro/in vivo correlations were performed (50 retrospective, 150 prospective). Overall predictive accuracy of the MP-assay was 86%, with correct indication of resistance in 94.5% and of sensitivity in 75.8% (P < 0.001). The results show that the proposed assay is capable of estimating the response probability of cytostatic drugs in individual tumours and thus can contribute to reducing the applications of non-effective drugs and, within limitations, to improving the basis of drug selection.

Antineoplastic Agents↗

Endocrine markers in malignant tumor cells producing parathyroid hormone-related protein.

Humoral hypercalcemia of malignancy may reflect the synthesis and secretion of biologically active parathyroid hormone-related protein (PTHrP) by a given tumor. In the present study we investigated 25 human non-endocrine carcinomas which were clinically associated with hypercalcemia (Ca > 11 mg%). By applying PTHrP-specific immunocytochemistry, PTHrP could be detected in all tumors. The intra-tumorous distribution was heterogeneous with strong positivity in relatively few cells or weak positivity in the majority of cells. Surprisingly, in the PTHrP producing cells none of the marker proteins typical of endocrine cells (neuron-specific enolase, Leu-7 antigen, chromogranin, synaptophysin and endocrine granule constituent) was found. On the other hand, PTHrP producing cells of endocrine origin, such as medullary cancer, or normal and adenomatous parathyroid glands, all produce these endocrine markers. Thus for the first time, the existence of peptide hormone producing tumor cells is reported without expression of endocrine markers. This indicates a special mechanism of PTHrP secretion.

Adenoma↗

[Androgen regulation of secreted growth factors in prostate carcinoma cell and tumor lines].

Previous studies have shown that part of steroid hormone action on hormone dependent carcinoma cells is mediated through secreted autocrine and paracrine growth factors. Coculture experiments using the androgen receptor positive human prostate carcinoma cell line LNCaP as feeder cells and the androgen receptor negative prostate cell line DU 145 as indicator cells, such as experiments with conditioned medium suggest that androgens might regulate proliferation of prostate carcinoma through a similar mechanism. LNCaP and DU 145 cells express high affinity EGF-receptors and show an increased growth rate under treatment with EGF, TGF alpha and FGF. The growth stimulating potential of LNCaP-conditioned medium can be enhanced by androgens. The polyanionic compounds suramin and dextran sulfates which have been shown to inactivate a variety of growth factors e.g. EGF/TGF alpha inhibit growth of LNCaP cells and DU 145 cells in a dose dependent and reversible fashion. Growth stimulation of LNCaP cells by EGF/TGF alpha can be completely reversed by simultaneous addition of polyanions but they inhibit androgen stimulation only partially. These data suggest the existence of at least two different mechanisms of growth regulation by androgens which can be distinguished by their different sensitivity of prostatic carcinoma cells to growth factor inhibitory agents. In order to investigate the therapeutic potential of these substances in complex, heterogeneous cell systems of solid tumors we treated 8 representative human prostate cancer lines in the nude mouse model. Systemic applications of polyanions revealed significant growth inhibition in hormone dependent as well as hormone independent xenografts. In androgen responsive lines growth inhibition was intensified by additional androgen withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens↗

Treatment results obtained in metastatic soft-tissue sarcoma with a combination of doxorubicin and dacarbazine or doxorubicin and ifosfamide.

Between 1982 and 1986, 38 patients with soft-tissue sarcomas were treated with the combination doxorubicin/dacarbazine (group A); between 1986 and 1990, another 46 patients received doxorubicin/ifosfamide (group B); and between 1990 and 1991, 11 patients received an alternating regimen of doxorubicin and ifosfamide (group C). The patients' characteristics were comparable among the three groups. Overall remission rates were 34% in group A, 44% in group B, and 18% in group C. The duration of remission was 10 months and the median survival was 13 months in groups A and B. Toxicity, especially myelotoxicity, was severe, with no marked differences being noted between the groups. We conclude that both doxorubicin/dacarbazine and doxorubicin/ifosfamide are active regimens in metastatic soft-tissue sarcomas; nevertheless, the overall prognosis for these patients remains poor.

Adolescent↗

Stereoisomers of calcium antagonists which differ markedly in their potencies as calcium blockers are equally effective in modulating drug transport by P-glycoprotein.

The (-)-isomer of verapamil is 10-fold more potent as a calcium antagonist than the (+)-isomer. However, both enantiomers are equally effective in increasing cellular accumulation of anticancer drugs [Gruber et al., Int J Cancer 41: 224-226, 1988]. In addition to verapamil, there exists a wide variety of stereoisomers with phenylalkylamines and dihydropyridine structures which markedly differ in their potency as calcium antagonists. We have tested these drugs for their ability to increase intracellular accumulation of [3H]vinblastine ([3H]VBL) in a doxorubicin-resistant cell line (F4-6RADR) derived from the Friend mouse leukemia cell line (F4-6P) and in COS-7 monkey kidney cells. Both cell types express substantial amounts of multidrug resistance gene 1 mRNA and P-glycoprotein as revealed by RNA and immuno blot analysis. The enantiomers with phenylalkylamine structures [(+/-)-verapamil; (+/-)-devapamil; (+/-)-emopamil)] and with dihydropyridine structures [(+/-)-isradipine; (+/-)-nimodipine; (+/-)-felodipine; (+/-)-nitrendipine; (+/-)-niguldipine] increased [3H]VBL accumulation in both cell lines at micromolar concentrations. Although the stereoisomers of these drugs differ markedly in their potency as calcium channel blockers they were about equally effective in increasing VBL levels in the cells. There was no substantial difference in the potencies of the phenylalkylamine drugs in affecting cellular [3H]VBL transport. Major potency differences, however, were observed in the dihydropyridine drug series with the niguldipine isomers as the most effective drugs. Moreover, the niguldipine enantiomers were equally as effective in reversing VBL resistance in F4-6RADR cells as were the verapamil enantiomers. Since (-)-niguldipine (B859-35) displays a 45-fold lower affinity for calcium channel binding sites than (+)-niguldipine, but is equally potent in inhibiting drug transport by P-glycoprotein and in reversing drug resistance, it may be, in addition to (+)-verapamil, another useful candidate drug for the treatment of multidrug resistance in cancer patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

DNA cytophotometry and prognosis in ovarian tumors of borderline malignancy. A clinicomorphologic study of 80 cases.

Surgical specimens of 80 ovarian tumors of borderline malignancy (OTBM) were investigated by scanning DNA cytophotometry. Diploid or euploid DNA histograms were found for 21 tumors, whereas 59 OTBM showed noneuploid or aneuploid DNA patterns. All patients were followed-up after surgery for at least 3 years (mean observation period, 6.7 years). Follow-up showed 11 cases of recurrent disease and 6 deaths. DNA findings and several other morphologic and clinical details (including patient age, histologic type and stage of disease, and extent of therapy) were correlated to the postoperative course. Statistical analyses disclosed that, of these parameters, only DNA content significantly affected prognosis. Recurrences and deaths resulting from tumor exclusively were observed among patients with noneuploid or aneuploid OTBM, whereas none of the diploid or euploid tumors recurred (P less than 0.05). DNA cytophotometry thus might be regarded as an effective complementary means to assess the prognosis of individual OTBM cases.

Adult↗

Karyotype instability and altered differentiation of rat sarcoma cells after retroviral infection.

The karyotypic and phenotypic stability of cultured rat fibrosarcoma cells was challenged by infection with Moloney murine sarcoma virus (MoMuSV). After transformation, the spindle-like morphology of the parental HH-16 cl.2/1 cells had altered to a rounded phenotype, which was maintained in tumors produced by inoculating transformed cells into congenic animals. In contrast to the parental cells, transformed cells lacked cables of cytokeratins 14-16 and 19 and showed reduction of the mesenchymal marker protein vimentin. Additionally, the morphologically altered cell clones tf-1 to tf-3 had lost growth arrest in the presence of dexamethasone. The DNA of the transformed cells contained between four and six randomly integrated proviral copies. Karyotypic alterations were manifested by reduction of morphologically intact chromosomes in the MoMuSV-transformed cells together with increase of structural aberrations. Three additional markers were identified in the virus-transformed cell clones. Karyotypic instability induced by MoMuSV infection appeared closely related to reduction of the cellular differentiation status, although only cells of clone tf-1 had increased metastatic potential.

Age Factors↗

[EBV-lymphadenitis: diagnostic possibilities using immunohistochemistry and in situ hybridization].

EBV infections were studied in paraffin embedded lymph node specimens (n = 20) on DNA, protein, and histological level. The lymph nodes were obtained from 20 patients all testing positive for EBV serologically. The EBV DNA was detected by non radioactive in-situ-hybridisation using the BamHI W fragment of the virus. The EBV latent membrane protein was detected immunohistochemically using the LMP antibody (DAKO). Infected cells were characterized by monoclonal B cell antibodies. By in-situ-hybridization we found positive signals for EBV in all specimens. In contrast, no LMP was detectable immunohistochemically in 2/20 lymph nodes.

Antibodies, Monoclonal↗

What's new in cytostatic drug resistance and pathology.

A major problem in cytostatic treatment of malignant tumors is the development of chemoresistant cell clones. An increased understanding of chemoresistance related mechanisms, improved methods for the detection and localization of resistant cell populations including predictive conclusions on the effectiveness of cytostatic drugs would contribute to the advancement of anti-tumor strategies. This paper reviews current concepts suggested for the development of cellular resistance to natural product drugs (anthracyclines, Vinca alkaloids, epipodophyllotoxines, antibiotics; so-called multidrug resistance substances), alkylating agents (nitrosureas, busulfan and mitomycin C), heavy metal compounds (cisplatin) and antifolates (5-fluorouracil, methotrexate) and describes the role of drug transporting and binding proteins (P-170-glycoprotein), detoxifying enzymes (glutathion-S-transferase, dihydrofolate reductase), DNA repair enzymes (topoisomerase I and II, polymerase alpha and beta), and genomic alterations (amplification, double minutes and homogeneous staining regions) due to resistance. It is focussed on the employment of morphological methods (light microscopy, immunocytochemistry, electron microscopy, fluorescence analysis, in situ hybridization, computer aided morphometric analysis) which will help to detect resistant cell clones in tumor biopsies. First correlations between histological data and clinical course will be reported. In the future, the morphological determination of chemoresistance may play an important role in applied functional tumor pathology.

Alkylating Agents↗

Induction of transforming growth factor beta by the antiestrogens droloxifene, tamoxifen, and toremifene in MCF-7 cells.

We have previously shown that transforming growth factor beta (TGF beta) is a hormonally regulated negative growth factor in estrogen responsive MCF-7 human breast cancer cells. We have now compared the antiestrogens tamoxifen, droloxifene (3-hydroxytamoxifen), and toremifene in their ability to induce the secretion of autoinhibitory TGF beta by MCF-7 cells. The main results are as follows: induction of TGF beta secretion by droloxifene is about two to three times higher than by identical concentrations of tamoxifen or toremifene. A 5-10 times higher concentration of tamoxifen or toremifene than droloxifene is necessary to reach a similar induction of TGF beta secretion. In contrast to tamoxifen, intermittent application of droloxifene is as effective as continuous treatment in inducing TGF beta secretion. We conclude from these data that TGF beta proteins represent markers of antiestrogen action and might also play a pivotal role in their mechanism of action. Droloxifene is a more effective inducer of TGF beta and a more potent growth inhibitor for estrogen responsive human breast cancer cells than tamoxifen and toremifene in vitro. Therefore, droloxifene might also possess a higher antiestrogenic potential in treatment of human breast cancer.

Antineoplastic Agents↗

Calcitriol decreases calcitonin secretion from a human medullary carcinoma cell line via specific receptor action.

Since calcitonin stimulates calcitriol production we examined whether a feedback regulatory mechanism exists between these hormones. First, the calcitriol receptor was localized immunocytochemically in cultured TT cells originating from a human thyroid medullary (C-cell) carcinoma. The receptor was demonstrated by light and electron microscopy in cell nuclei and in small amounts in the cytosol. Receptor levels increased when the cells were cultured at physiological (120-240 pmol/l) or somewhat higher concentrations (1200-2400 pmol/l) of calcitriol. In parallel, the same doses of calcitriol markedly inhibited secretion of calcitonin into the medium. These results indicate a feedback loop between calcitriol and calcitonin.

Calbindins↗

Membrane vesicle formation due to acquired mitoxantrone resistance in human gastric carcinoma cell line EPG85-257.

A newly established gastric carcinoma cell line (EPG85-257P) exhibited a high sensitivity to mitoxantrone (DHAD) as determined by a monolayer proliferation assay. The concentration to inhibit cell growth to 50% of controls (IC50) was 0.0022 micrograms/ml culture medium. The cells were continuously incubated for more than 4 months in the presence of stepwise increased concentrations of DHAD, and the IC50 was increased to 0.41 micrograms/ml, i.e., 186.4-fold. This resistant variant was named EPG85-257RNOV. The EPG85-257RNOV cells became cross-resistant to Adriamycin with enhanced IC50 by 10.5-fold and to daunomycin with enhanced IC50 by 3.9-fold. No distinct resistance was observed to vinblastine, vincristine, and colchicine. Verapamil (10(-6), 4 X 10(-6) and 10(-5) M) and cyclosporin A (10(-6), 3 X 10(-6) and 10(-5) M) did not reverse DHAD resistance. As shown by immunocytochemistry (monoclonal antibodies: C219 and JSB-1) and Northern blot analysis, DHAD resistance was not associated with the appearance of the multidrug resistance (MDR)-associated (Mr 170,000) P-glycoprotein or the overexpression of P-glycoprotein mRNA. The data indicate a chemoresistance pattern unlike typical MDR (often called "atypical" MDR). The phenotypes of parent and resistant EPG85-257 cells were compared using interference contrast microscopy, electron microscopy, and immunocytochemistry. After DHAD application the following structural characteristics were found to be associated with emergence of resistance: (a) intensive formation of surface vesicles in the resistant variant. Such vesicles were almost absent in sensitive cells; (b) the vesicles contained the selecting DHAD which was visualized by its blue color; and (c) in electron microscopy the vesicles were formed by an inner and an outer double membrane, presumably derived from the plasmalemma. These observations suggest a complex cellular mechanism responsible for DHAD resistance which includes formation of membrane vesicles, vesicular drug binding, and drug compartmentalization.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Nicotinamide and nicotinamide analogues as antitumor promoters in mouse skin.

Phorbol ester-induced promotion of initiated NMRI mouse skin keratinocytes to papillomas could be largely prevented when nicotinamide-like inhibitors of poly(ADP-ribose)polymerase (nicotinamide, benzamide, 3-aminobenzamide) were applied simultaneously with 12-O-tetradecanoylphorbol-13-acetate (TPA). A similar suppression of tumor promotion by nicotinamide analogues was demonstrated in clone 41 JB6 epidermal cells which are promotable by TPA to anchorage-independent growth. The antipromotion effect of nicotinamide analogues, however, does not appear to come about by an inhibition of poly(ADP-ribose)polymerase. Acid analogues of nicotinamide, such as benzoic acid or 3-aminobenzoic acid which do not inhibit the polymerase, showed antipromotion activity similar to that of their corresponding amides. It could also be ruled out that these antipromoters mediate their effect on keratinocytes by a cytostatic action, by scavenging the promoter TPA in a chemical reaction, or by inhibiting protein kinase C. In initiated mouse skin, nicotinamide analogues strongly suppressed TPA-induced accumulation of inflammatory cells and vascular permeability, while epidermal hyperplasia was not significantly affected.

9,10-Dimethyl-1,2-benzanthracene↗