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Studies on type I collagen in skin fibroblasts cultured from twins with lethal osteogenesis imperfecta.

100%
Galicka A., Wołczyński S., Gindzieński A.
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2003
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vol. 50
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issue 2
481-488
EN
Studies on type I procollagen produced by skin fibroblasts cultured from twins with lethal type II of osteogenesis imperfecta (OI) showed that biosynthesis of collagen (measured by L-[5-3H]proline incorporation into proteins susceptible to the action of bacterial collagenase) was slightly increased as compared to the control healthy infant. SDS/PAGE showed that the fibroblasts synthesized and secreted only normal type I procollagen. Electrophoretic analysis of collagen chains and CNBr peptides showed the same pattern of electrophoretic migration as in the controls. The lack of posttranslational overmodification of the collagen molecule suggested a molecular defect near the amino terminus of the collagen helix. Digestion of OI type I collagen with trypsin at 30°C for 5 min generated a shorter than normal α2 chain which melted at 36°C. Direct sequencing of an asymmetric PCR product revealed a heterozygous single nucleotide change C→G causing a substitution of histidine by aspartic acid in the α2 chain at position 92. Pericellular processing of type I procollagen by the twin's fibroblasts yielded a later appearance of the intermediate pC-α1(I) form as compared with control cells.
2
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Can transforming growth factor-β1 and retinoids modify the activity of estradiol and antiestrogens in MCF-7 breast cancer cells?

88%
Czeczuga-Semeniuk E., Anchim T., Dzięcioł J., Dąbrowska M., Wołczyński S.
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vol. 51
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issue 3
733-745
EN
Retinoic acid and transforming growth factor-β (TGF-β) affect differentiation, proliferation and carcinogenesis of epithelial cells. The effect of both compounds on the proliferation of cells of the hormone sensitive human breast cancer cell line (ER+) MCF-7 was assessed in the presence of estradiol and tamoxifen. The assay was based on [3H]thymidine incorporation and the proliferative activity of PCNA- and Ki 67-positive cells. The apoptotic index and expression of the Bcl-2 and p53 antigens in MCF-7 cells were also determined. Exogenous TGF-β1 added to the cell culture showed antiproliferative activity within the concentration range of 0.003-30 ng/ml. Irrespective of TGF-β1 concentrations, a marked reduction in the stimulatory action of estradiol (10-9 and 10-8 M) was observed whereas in combination with tamoxifen (10-7 and 10-6 M) only 30 ng/ml TGF-β1 caused a statistically significant reduction to aproximately 30% of the proliferative cells. In further experiments we examined the effect of exposure of breast cancer cells to retinoids in combination with TGF-β1. The incorporation of [3H]thymidine into MCF-7 cells was inhibited to 52 ± 19% (control =100%) by 3 ng/ml TGF-β1, and this dose was used throughout. It was found that addition of TGF-β1 and isotretinoin to the culture did not decrease proliferation, while TGF-β1 and tretinoin at low concentrations (3 × 10-8 and 3 × 10-7 M) reduced the percentage of proliferating cells by aproximately 30% (67±8% and 67±5%, P <0.05 compared to values in the tretinoin group). Both retinoids also led to a statistically significant decrease in the stimulatory effect of 10-9 M estradiol, attenuated by TGF-β1. In addition, the retinoids in combination with TGF-β1 and tamoxifen (10-6 M) caused a further reduction in the percentage of proliferating cells. Immunocytochemical analysis showed that all the examined compounds gave a statistically significant reduction in the percentage of cells with a positive reaction to PCNA and Ki 67 antigen. TGF-β1, isotretinoin and tretinoin added to the culture resulted in the lowest percentage of PCNA positive cells. However, the lowest fraction of Ki 67 positive cells was observed after addition of isotretinoin. The obtained results also confirm the fact that the well-known regulatory proteins Bcl-2 and p53 play an important role in the regulation of apoptosis in the MCF-7 cell line, with lowered Bcl-2 expression accompanying easier apoptotic induction. The majority of the examined compounds act via the p53 pathway although some bypass this important proapoptotic factor.
3
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Molecular modelling, synthesis and antitumour activity of carbocyclic analogues of netropsin and distamycin - new carriers of alkylating elements.

88%
Bartulewicz D., Markowska A., Wołczyński S., Dąbrowska M., Różański A.
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2000
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vol. 47
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issue 1
23-35
EN
A series of netropsin and distamycin analogues was synthesised and investigated by molecular modelling. The lowest-energy conformations of four carbocyclic lexitropsins, potential carriers of alkylating elements, were obtained using the HyperChem 4.0 program, and compared with the DNA-lexitropsin crystal structures from the Brookhaven National Laboratory Protein Data Bank. A method for synthesis of carbocyclic lexitropsins was elaborated, with the use of a nitro group or azobenzene as precursors for the aromatic amino group. The influence of methoxy group in ortho position with respect to amide groups on the activity of the new compounds was investigated. All of the compounds tested showed high antitumour activity in the standard cell line of mammalian tumour MCF-7.
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