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Biomedical subjects

G Bernhardt

Publications and source records attributed to G Bernhardt.

At least 55 records · Page 3Linked to original sources
PubMed1995

Development of a parenterally administrable hydrosol preparation of the "third generation platinum complex" [(+/-)-1,2-bis(4-fluorophenyl)ethylenediamine]dichloroplatinum(II). Part 1. Preparation and studies on the stability and antitumor activity.

The development of a galenical formulation for poorly water soluble dichloroplatinum(II) complexes suitable for the parenteral administration in cancer chemotherapy is described. The procedure, which we elaborated for [(+/-)-1,2-bis(4-fluorophenyl)ethylenediamine]dichloroplatinum(II) (rac-4F-PtCl2), is based on the reaction of a soluble diaquaplatinum(II) salt with sodium chloride in water in the presence of pluronic F 68 as stabilizer and results in a sufficiently stable colloidal solution (i.e. hydrosol). In contrast to the poorly water soluble synthetic rac-4F-PtCl2, which was ineffective towards the hormone sensitive MXT-M-3.2 breast cancer of the mouse, its hydrosol formulation proved to be highly active and was very well tolerated.

Animals↗
PubMed1995

Hyaluronidase significantly enhances the efficacy of regional vinblastine chemotherapy of malignant melanoma.

The regional chemotherapy of the human malignant melanomas (SK-MEL-2, -3, -5, -24) implanted in NMRI nu/nu mice with a combination of the hyaluronic-acid-cleaving enzyme hyaluronidase (HYase) and vinblastine is a very effective therapeutic procedure. In three out of four melanoma models (SK-MEL-2, -3, -5) the weekly peritumoral administration of high-dose HYase (100,000 IU/kg) 4 h prior to the injection of 0.3 mg/kg vinblastine in the vicinity of the tumor (seven weekly therapeutic cycles) caused marked antitumor effects, while HYase and vinblastine were inactive when given alone. The pretreatment with HYase, which is well tolerated by the test animals, prevented local inflammation reactions commonly seen after subcutaneous vinblastine administration. Tumor growth and metastatic behavior of the melanomas used were neither increased nor reduced by HYase after peritumoral administration without subsequent vinblastine injection. The curative activity of the regional chemotherapy with HYase/vinblastine could be demonstrated on the SK-Mel-3 melanoma. After an observation time of 18 weeks, tumor cells could no longer be detected in the subcutaneous region of the former lesion. Only macrophages, which had abundantly incorporated melanin, gave evidence of previously growing tumors. In contrast to the controls, no metastases could be observed in the axillary lymph nodes of the test animals.

Animals↗
PubMed1994

Interaction of poliovirus with its cell surface binding site.

The interaction of poliovirus with its cellular binding sites was characterized by using a receptor-excess silicon oil partition assay. Poliovirus type 1 Mahoney [PV1(M)] binding to HeLa cells fits a theoretical simple bimolecular noncooperative binding curve with an equilibrium dissociation constant (Kd) of 4.3 x 10(7) cells.ml-1 at 4 degrees, or 2.1 x 10(-10) M, assuming 3000 virus binding sites/cell. The association rate of complex formation was measured to be 3.6 x 10(-9) ml.cell-1.min-1 (7.2 x 10(8) M-1.min-1) and the dissociation rate calculated to be 1.5 x 10(-1) min-1, giving the complex a half-life of 4.5 min. The equilibrium dissociation constant, association rate, and dissociation rate were also measured for the binding of the attenuated poliovirus type 3 Sabin strain [PV3(S)] to HeLa cells. PV3(S) bound HeLa cells with a Kd of 3.3 x 10(7) cells.ml-1 (1.6 x 10(-10) M), an association rate of 4.1 x 10(-9) ml.cell-1.min-1 (8.2 x 10(8) M-1.min-1), and a dissociation rate calculated to be 1.4 x 10(-1) min-1, giving the complex a half-life of 5.1 min. Thus the virulent and avirulent strains of poliovirus bind HeLa cells with nearly identical binding constants and rate constants. Equilibrium binding constants for PV1(M) to various other cell types varied from a high affinity of 4.1 x 10(6) cells.ml-1 for JA-1 cells to a low affinity of 7 x 10(7) cells.ml-1 for NGP cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Molecular characterization of the cellular receptor for poliovirus.

The expression of the human poliovirus receptor (hPVR) in several cultured cell lines was studied with the use of different antibodies directed against hPVR proteins. Immunoprecipitations of metabolically labeled cell lysates revealed that membrane-bound glycoforms of hPVR proteins have a molecular weight of about 80 kDa. By applying inhibitors of the glycosylation pathway (deoxymannojirimycin and swainsonine) we were able to monitor the modification of the hPVR glycoproteins when passing through the processing pathway. We show that a 67-kDa hPVR protein identified earlier (using a vaccinia virus expression system) is an intermediate glycoform probably located in the endoplasmic reticulum or cis-Golgi. Further modification of this glycoform is blocked by vaccinia virus infection or by the inhibitor deoxymannojirimycin. We, therefore, conclude that the 80-kDa glycoforms identified here are the fully processed hPVR isoforms. Surface iodination confirms that only the 80-kDa glycoforms are expressed on the cell surface. Treatment with various deglycosylating enzymes (N-Glycanase, O-Glycanase, Endo-H, and neuraminidase) demonstrates that the hPVR proteins bear sialylated complex-type oligosaccharides. Deglycosylation of the hPVR proteins also reveals the presence of both hPVR membrane-bound forms in cultured cells. Their relative expression levels, with respect to each other, vary considerably. The distribution of these hPVR isoforms in tissues may help explain the natural function of the hPVR proteins.

Animals↗
PubMed1994

The poliovirus receptor: identification of domains and amino acid residues critical for virus binding.

The N-terminal domain 1 of the human poliovirus receptor (hPVR), a three-domain, immunoglobulin-like molecule, was previously shown to be necessary and sufficient to confer poliovirus (PV) susceptibility to mouse cells. However, studies with truncated versions of hPVR suggested that the C-terminal hPVR domains may contribute to receptor function. We describe sets of hybrid receptors, constructed between hPVR and hICAM-1 (human intercellular adhesion molecule-1) that were tested in mouse cells for hPVR functionally. Whereas the context in which hPVR is expressed is of minor importance, all three domains of hPVR are required to reach wild-type function. Single and multiple amino acid exchanges were introduced into the first hPVR domain in order to localize regions that were involved in virus-receptor interactions. The mutations were analyzed for their ability to bind PV1 (Mahoney) or monoclonal antibodies as well as their ability to support viral replication in either the hPVR alpha or hybrid hPVR-hICAM-1 receptor context. When placed into a model of the V domain of hPVR, the effect of the mutations indicated that the C'C"D as well as the DE region harbored amino acids that contacted the PV1(M) surface in the process of receptor-virus complex formation. The binding of the virus to the receptor and subsequent uptake into the cells were linked; no hPVR mutants were observed that bound the virus but blocked infection. N-glycosylation of the four sites in domains 1 and 2 is not required for hPVR function, but glycosylation in domain 1 has a greater effect on receptor function than that of domain 2.

Animals↗
PubMed1994

In vitro activity of immunoconjugates between cisplatin and an anti-CA125 monoclonal antibody on ovarian cancer cell lines.

Cis-diammine dichloro platinum (II) (CDDP), is a highly potent antineoplastic agent that is used in the treatment of ovarian cancer. However, the clinical use of CDDP is restricted by its severe side effects. In order to reduce these side effects and to enhance its therapeutic efficacy, we developed specific immunoconjugates consisting of the murine monoclonal antibody OC125 and CDDP, using diethylene triamine pentaacetic acid (DTPA) as a linker. The coupling efficiencies of the different preparations synthesized, varied between 1.10 +/- 0.42 and 2.65 +/- 1.60 mol of CDDP per mol of antibody protein. Despite the chemical modification of the antibody molecule, specific binding activity of the OC125-CDDP conjugates toward the CA125 antigen was maintained as was demonstrated by means of immunohisto-/cytochemical staining of frozen sections of ovarian cancer tissue, amniotic epithelium, and the CA125 positive ovarian cancer cell line NIH:OVCAR 3. The antiproliferative activity of the immunoconjugates was tested against the human ovarian cancer cell lines NIH:OVCAR 3 and SKOV 3, applying a kinetic crystal violet microassay. Despite the promising results obtained with the specific immunostaining of the target cells, no significant antiproliferative activity of our immunoconjugates against the cell lines tested was observed. One possible explanation for the lack of antitumor activity could be the fact that CA125 is released in large amounts by the NIH:OVCAR 3 cells. This may have prevented an efficient immunotargeting of the cancer cells by the formation of soluble immune complexes.

Antibodies, Monoclonal↗
PubMed1994

Cleavage site of the poliovirus receptor signal sequence.

We have shown recently that the human poliovirus receptors (hPVRs) expressed on the surface of cultured cells are 80K glycoproteins, whereas the previously reported 67K forms are partially glycosylated intermediate glycoforms. Both the membrane-bound 80K and 67K forms of hPVR are glycosylated derivatives of the two isoforms hPVR alpha and hPVR delta, where the latter two can be resolved only by SDS-PAGE upon enzymatic deglycosylation. Here we report the N-terminal sequence analysis of the mature 80K as well as the intermediate 67K glycoforms of hPVR which has allowed us to identify the signal peptidase cleavage site of the unprocessed hPVR. The signal sequence that directs translocation of hPVR across the membrane of the endoplasmic reticulum on its route to the glycoprocessing pathway has thus been defined. We compare this signal sequence with those of the putative monkey poliovirus receptor and the mouse poliovirus receptor homologue.

Amino Acid Sequence↗
PubMed1994

The human poliovirus receptor alpha is a serine phosphoprotein.

The human receptors for poliovirus (hPVR) are members of the immunoglobulin superfamily. Whereas the two membrane-bound isoforms, hPVR alpha and hPVR delta, share identical three-domain extracellular portions, their C-terminal cytoplasmic parts differ considerably. This feature is well conserved in the corresponding monkey proteins AGM alpha 1, AGM delta 1, and AGM alpha 2. The cellular function of these proteins is presently unknown. In this short communication we report that hPVR alpha and possibly also AGM alpha 1 and AGM alpha 2, but not the delta isoforms, are phosphoproteins. The phosphorylation occurs at a serine in the cytoplasmic tails of these receptors. We further present evidence suggesting that the kinase responsible for the phosphorylation is calcium/calmodulin kinase II.

Amino Acid Sequence↗
PubMed1993

Antitumour activity of miltefosine alone and after combination with platinum complexes on MXT mouse mammary carcinoma models.

Miltefosine, an alkylphosphocholine structurally related to alkyllysophospholipids showed highly selective antitumour activity against the hormone-sensitive variant of the s.c. transplantable MXT mouse mammary adenocarcinoma, the ovary-dependent MXT (M3.2), whereas it was inactive against the hormone-insensitive MXT (M3.2) OVEX variant. A dose of 32 mg/kg miltefosine p.o. daily for 5 weeks was well tolerated. Histopathological evaluation gave no signs of gastroenteral toxicity. After therapy the microarchitecture of the MXT (M3.2) tumours changed from that of a moderately differentiated adenocarcinoma to that of an anaplastic mammary carcinoma. A dose of 16 mg/kg miltefosine p.o. daily, though in effective per se, enhanced the antitumour activity of suboptimal i.p. doses of cisplatin and the hormone-like platinum analogue meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl) ethylenediamine]dichloroplatinum(II). Furthermore, it was shown, that miltefosine exhibited no (anti)hormonal properties. However, the mechanism of action of miltefosine remains unclear.

Adenocarcinoma↗
PubMed1992

Dichloro-[1-(hydroxyphenyl)-2-phenylethylenediamine]platinum(II) complexes: testing on the human ovarian cancer cell lines NIH: OVCAR3 and SK OV 3.

The diastereoisomeric dichloro-[1-(2-, 3- and 4-hydroxyphenyl)-2-phenylethylenediamine]platinum(II) complexes were tested on two human ovarian cancer cell lines NIH: OVCAR-3 and SK-OV-3, both resistant against cisplatin. Dichloro-[threo-1-(3-hydroxyphenyl)-2-phenylethylenediamine]platinum(II) (threo-5-PtCl2) proved to be the most active representative of the new series, producing cytocidal effects at a concentration range of 2.5 to 5.0 microM on the NIH: OVCAR-3 cell line. On the more resistant SK-OV-3 cell line, threo-5-PtCl2 was only moderately active, while in combination with BSO, a GSH level lowering compound, threo-5-PtCl2 showed a strong synergistic effect.

Adenocarcinoma↗
PubMed1992

Chemosensitivity of human MCF-7 breast cancer cells to diastereoisomeric diaqua(1,2-diphenylethylenediamine) platinum(II) sulfates and specific platinum accumulation.

Cisplatin, raceme-diaqua[1,2-bis(4-fluorophenyl)ethylenediamine]platinu m(II) sulfate (compound I), meso-diaqua[1,2-bis(4-fluorophenyl)ethylenediamine]-platinum(II) sulfate (compound II), and meso-diaqua[1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]platinum(II) sulfate (compound III) were compared with regard to their effect on the MCF-7 breast cancer cell line in vitro. At equimolar concentrations (5 microM), cisplatin, compound I, and compound II were equiactive after 231 h drug exposure, whereas compound III was ineffective. Although compounds I and II showed markedly greater inactivation than did cisplatin after 6 h incubation with culture medium, compound I (but not compound II) exhibited antitumor activity equivalent to that of cisplatin when cells were exposed to the drugs for 6 h. Platinum measurements by neutron-activation analysis revealed that compound I was selectively and rapidly accumulated by MCF-7 cells, resulting in a high degree of DNA platination within the first few hours of drug exposure. However, when the drug-exposure period was long enough, platinum enrichment was not reflected in an overall difference in the cytotoxicity of compound I vs cisplatin. Nevertheless, compound I should be superior to cisplatin in vivo, provided that effective plasma levels can be maintained for about 6 h.

Breast Neoplasms↗
PubMed1992

Standardized kinetic microassay to quantify differential chemosensitivity on the basis of proliferative activity.

Conventionally in vitro cytotoxicity assays are performed as single-end-point determinations. To compensate for the diversity of growth rates among different cell lines in this report we describe a computerized kinetic chemosensitivity assay based on quantification of biomass by staining cells with crystal violet. As a prerequisite four human breast cancer cell lines (MDA-MB-231, MCF-7, T-47-D and ZR-75-1) were characterized with regard to oestrogen and progesterone receptor content, modal chromosome number and proliferation kinetics depending on the number of passages in culture. With prolonged time in culture for ZR-75-1 exposed to various concentrations of cisplatinum a dose-related increase in drug effect was observed. Owing to a correction of the T/C values for the initial cell mass (at the time when drug is added) a sharp distinction between cytostatic and cytocidal drug effects becomes obvious in plots of corrected T/C values versus time of incubation. The influence of the untreated control on the corrected T/C values and possible time courses of theoretical inhibition profiles (reflecting cytostatic, transient cytotoxic or cytocidal drug effects as well as development of resistance) and their relationship to the corresponding growth curves of drug-treated cells are discussed. Chemosensitivity assays with diethylstilbestrol dipropionate, tamoxifen, melphalan, cisplatinum, vinblastine, Adriamycin and 5-fluorouracil prove the theoretical considerations to be true for MDA-MB-231, MCF-7, T-47-D and ZR-75-1 human breast cancer cell lines in practice.

Adenocarcinoma↗
PubMed1992

Hyaluronidase enhances the activity of adriamycin in breast cancer models in vitro and in vivo.

The effect of hyaluronidase and a combination of hyaluronidase with Adriamycin was investigated on several breast cancer models in vitro and in vivo. In vitro enzyme treatment (using concentrations up to 80,000 IU/1) of murine (MXT-, MXT +/-, and MXT+) and human (MCF-7, ZR-75-1 and T-47-D) breast cancer cell lines did not inhibit tumour cell proliferation (measured by a kinetic crystal violet assay) in either case. Although high-dose hyaluronidase (1.2 x 10(6) IU/kg) was ineffective, when administered peritumourally to the MXT M3.2 mammary carcinoma of the B6D2F1 mouse, it is remarkable that five "megadoses" were excellently tolerated. However, the antineoplastic activity of Adriamycin against the oestrogen-receptor-positive variant of the MXT tumour was significantly enhanced by combination with concentrations of hyaluronidase that were inactive per se, both in vitro and in vivo. Interestingly, the enhancement of the in vivo antitumour activity was not compromised by toxic side-effects.

Animals↗
PubMed1992

[1,2-Bis(2-hydroxyphenyl)ethylenediamine]dichloroplatinum(II), a new compound for the therapy of ovarian cancer. II. Synthesis and preliminary testing of the enantiomeric complexes.

The enantiomeric [1,2-bis(2-hydroxyphenyl)-ethylenediamine]dichloroplatinum(II) complexes were synthesized and their configuration assessed. A preliminary test in the cisplatin-resistant human NIH:OVCAR-3 ovarian cancer cell line, which was previously characterized by its sensitivity against several therapeutically used drugs, showed that both enantiomers produce cytocidal effects in a concentration of 2.5 microM. A difference between the enantiomers became evident from the faster onset of cytocidal activity of the S,S-configurated compound.

Adenocarcinoma↗
PubMed1992

[1,2-Bis(2-hydroxyphenyl)ethylenediamine]dichloroplatinum(II), a new compound for the therapy of ovarian cancer. III. Detailed evaluation of the antitumor activity of the enantiomeric complexes on the human NIH:OVCAR-3 ovarian cancer cell line.

The stereoisomeric [1,2-bis(2-hydroxyphenyl)ethylenediamine]dichloroplatinum(II) complexes were thoroughly tested on the cisplatin-resistant human NIH:OVCAR-3 ovarian cancer cell line. The racemate and its enantiomers produced cytocidal effects at a concentration of 2.5 microM (incubation time 256 h). The meso form, however, was merely cytostatically active. Differences between the enantiomers became evident after a short drug incubation time (1 h) followed by an incubation in drug-free medium (243 h). The S,S-configurated enantiomer (-)-3-PtCl2 proved to be the most active compound. To achieve cytocidal effects concentrations of 2.5-5.0 microM and incubation times of about 3 h were necessary for (-)-3-PtCl2. This compound is also sufficiently stable under test conditions as shown by the preincubation in cell-free medium for 3 h. These results and the augmentation of its antitumor activity by buthionine sulfoximine recommend the further preclinical development of (-)-3-PtCl2 for clinical use.

Antineoplastic Agents↗
PubMed1992

Establishment and characterization of a human papillary thyroid carcinoma cell line with oxyphilic differentiation (ONCO-DG 1).

In the present study the establishment and characterization of a new oxyphilic papillary thyroid carcinoma cell line--ONCO-DG1- is given. With immunohistological, histochemical and flow cytometric methods, ONCO-DG 1 cells revealed features of epithelial differentiation. Furthermore the cells formed von Kossa-positive deposits resembling psammoma bodies in monolayer and spheroid culture until late passages. The tumor cell line is now in the 40th subculture. Because of the ability to form multicellular tumor spheroids (MCTS), this cell line is a good model for examining the interaction between thyroid tumor cells and confluent human endothelial cells on extracellular matrix in vitro. It is also suitable for xenotransplantation studies, because it is tumorigenic in NMRI nude mice in vivo.

Animals↗
PubMed1991

Different response of murine and human mammary tumour models to a series of diastereoisomeric [1,2-bis(difluorophenyl) ethylenediamine]dichloroplatinum(II) complexes.

A series of isomeric [1,2-bis(difluorophenyl) ethylenediamine]dichloroplatinum(II) complexes and cisplatin were tested on the P388 leukemia and on the murine hormone-independent MXT (M3.2) OVEX and the ovarian-hormone-dependent MXT (M3.2) mammary carcinoma for evaluating antineoplastic activity against breast cancer in vivo. Although these results were heterogeneous, a trend to the 2,6-difluorosubstituted compound as the most active platinum complex was observed. For the development of a large-scale in vitro screening method on human breast cancer cell lines, cell number, [3H]thymidine incorporation, and crystal violet staining were evaluated as parameters for end-point determination. Chemosensitivity testing on the human breast cancer cell lines MDA-MB-231 and MCF-7 unambiguously identified [1,2-bis(2,4-difluorophenyl)ethylenediamine]dichloroplatinum(II) as the complex with the highest activity in the crystal violet micro-assay. In equimolar concentration this compound was superior to cisplatin on both cell lines. The analysis of the conflicting results of this study indicates that murine mammary carcinomas are most probably unrealistic and inappropriate models for the screening of cytotoxic platinum complexes with potential activity on human breast cancer.

Adenocarcinoma↗