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Transformation by oncogenic ras-p21 alters the processing and subcellular localization of the lysosomal protease
M Démoz1, R Castino, A Dragonetti
1Department of Medical Sciences, A. Avogadro University, Novara, Italy.
Abstract:
The expression, processing, and intracellular localization of cathepsin D (CD), an endosomal-lysosomal protease involved in malignancy, were studied in rat embryo fibroblasts transformed with an active mutant of c-Ha-ras oncogene. The pattern of the processed molecular forms of CD, comprising two single-chain mature forms of 45 and 43 kDa and two double-chain mature forms of 34 + 9 kDa and 30 + 14 kDa, expressed by the parental cell line was similar to that found in normal rat liver cells. By contrast, in the ras-transfected counterpart this pattern was profoundly altered in that the 45 kDa species was much less represented and the 30 + 14 kDa species virtually absent. In both untransformed and ras-transformed cells the conversion of proCD into mature forms was not inhibited by ammonium chloride, which is known to increase the intravacuolar pH of post-Golgi compartments. Yet, this drug induced the accumulation of the 43 and 45 kDa molecular forms of mature CD in ras-transformed cells and of the 34 kDa molecule in untransformed cells. As compared to controls, in ras-transformed fibroblasts vacuolar compartments containing CD were reduced in number and mostly located toward the periphery of the cell. This contrasted with the perinuclear distribution of CD-positive granules in untransformed cells. Serum deprivation did not affect the growth, nor the intra- and extracellular accumulation of CD activity in ras-transformed cultures, while it blocked the growth and strongly stimulated the accumulation of CD in the medium in cultures of control fibroblasts. Altogether these data are indicative for a crucial role of ras GTPase in the regulation of the transport between post-Golgi organelles.
Insights
Ras oncogene transformation alters cathepsin D (CD) processing and localization in fibroblasts. These changes suggest ras GTPase critically regulates transport between post-Golgi organelles.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Cathepsin D (CD) is an endosomal-lysosomal protease implicated in cancer progression.
- The c-Ha-ras oncogene plays a role in cell signaling and transformation.
Purpose of the Study:
- To investigate the impact of ras oncogene transformation on cathepsin D expression, processing, and intracellular localization.
- To elucidate the role of ras GTPase in regulating post-Golgi organelle transport.
Main Methods:
- Analysis of cathepsin D molecular forms in ras-transfected and parental rat embryo fibroblasts.
- Treatment with ammonium chloride to assess pH-dependent processing.
- Microscopic examination of cathepsin D localization in different cell types.
- Evaluation of serum deprivation effects on cell growth and CD activity.
Main Results:
- Ras transformation altered the pattern of mature cathepsin D molecular forms, with reduced 45 kDa and absent 30+14 kDa species.
- Ammonium chloride induced accumulation of specific mature CD forms in both cell types but with distinct patterns.
- Ras-transformed cells showed fewer, peripherally located CD-positive vacuoles compared to perinuclear distribution in controls.
- Serum deprivation did not affect ras-transformed cell growth or CD accumulation, unlike control cells.
Conclusions:
- Ras oncogene significantly alters cathepsin D processing and intracellular trafficking.
- Ras GTPase appears to be a key regulator of transport dynamics within post-Golgi compartments.
- These findings highlight a potential role for CD dysregulation in ras-mediated oncogenesis.