Related Experiment Videos
Tyrosine protein kinase activity of human hyperplastic prostate and carcinoma cell lines PC3 and DU145
Y Durocher1, A Chapdelaine, S Chevalier
1Maisonneuve-Rosemont Research Center, Montreal, Quebec, Canada.
Abstract:
Using the substrate poly[Glu80Na,Tyr20] [poly(GT)] and the autoradiographic detection of alkali-resistant phosphoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tyrosine protein kinase (TPK) has been evidenced in human hyperplastic prostates (BPH) and the prostatic carcinoma cell lines PC3 and DU145. The enzyme was mainly found in the soluble fractions from hyperplastic tissues and in Triton extracts from the cell lines. However, its specific activity in tissues was 1.5- to 4.5-fold times higher in particulate than in soluble fractions and it was of the same order of magnitude as that of neoplastic cells. Under these conditions, no activity was detected in human seminal plasma and in sera from normal adult males or patients with BPH and/or prostatic carcinoma. On the other hand, some TPK activity was associated with human spermatozoa, with a specific activity 4- to 6-fold lower than in BPH tissue fractions and a total activity, per 10(6) cells, 5- to 20-fold lower than that in prostatic carcinoma cells. The activity of prostatic TPK was dependent upon the presence of the divalent cations Mn2+ or Mg2+ and it was completely abolished by heat denaturation. Angiotensin II, casein, and histone H2B were poor substrates compared to poly(GT). The TPK activities towards poly(GT) as well as endogenous proteins were not stimulated by epidermal growth factor and insulin or by dihydrotestosterone and estradiol. The autoradiography of alkali-resistant phosphoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several bands in both BPH tissues and neoplastic cells (molecular weight ranging from 17,000 to 200,000). Preliminary characterization of TPK by gel filtration on Sephacryl S-300 showed that the soluble enzyme from BPH tissues had a molecular weight of 50,000, while the particulate-associated TPK, when assayed on poly(GT), eluted with proteins of Mr 210,000. When these peak fractions were used for endogenous phosphorylation, several major alkali-resistant phosphoproteins in the range of Mr 40,000-60,000 were evidenced, together with a Mr 110,000 band phosphorylated by the particulate TPK of Mr 210,000. In similar conditions, the TPK solubilized from rat liver membranes and partially purified by gel filtration was associated with a Mr 170,000 alkali-resistant phosphoprotein. Thus, TPKs are expressed in BPH tissues and carcinoma cell lines. In BPH tissues, two forms of TPK are expressed and the predominant enzyme is soluble and of low molecular weight (Mr 40,000-60,000).
Insights
Tyrosine protein kinase (TPK) activity was detected in human benign prostatic hyperplasia (BPH) tissues and prostate cancer cell lines. This enzyme, crucial for cell signaling, was found in both soluble and particulate fractions, with higher specific activity in the latter.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Tyrosine protein kinases (TPKs) play critical roles in cellular signaling pathways.
- Aberrant TPK activity is implicated in the development and progression of various cancers, including prostate cancer.
- Understanding TPK expression and activity in benign prostatic hyperplasia (BPH) and prostate cancer is essential for therapeutic development.
Purpose of the Study:
- To investigate the presence and characteristics of tyrosine protein kinase (TPK) in human benign prostatic hyperplasia (BPH) tissues and prostate cancer cell lines (PC3, DU145).
- To compare TPK activity and localization in BPH tissues versus neoplastic cells.
- To explore the enzymatic properties, substrate specificity, and molecular weight of TPKs in these biological samples.
Main Methods:
- Utilized poly[Glu80Na,Tyr20] [poly(GT)] as a substrate for TPK detection.
- Employed autoradiographic detection of alkali-resistant phosphoproteins after SDS-PAGE.
- Fractionated tissue and cell extracts into soluble and particulate components for differential enzyme activity assessment.
- Performed gel filtration chromatography (Sephacryl S-300) for preliminary molecular weight characterization of TPKs.
Main Results:
- TPK activity was identified in human BPH tissues and PC3/DU145 prostate cancer cell lines.
- The enzyme was predominantly found in soluble fractions of BPH tissues and Triton extracts of cell lines.
- Specific TPK activity was higher in particulate fractions of BPH tissues compared to soluble fractions.
- No significant TPK activity was detected in seminal plasma or sera from normal individuals or cancer patients.
- TPK activity in human spermatozoa was significantly lower than in BPH tissue fractions.
- TPK activity was dependent on Mn2+ or Mg2+ and abolished by heat denaturation.
- Poly(GT) was a superior substrate compared to angiotensin II, casein, and histone H2B.
- TPK activity was not stimulated by EGF, insulin, dihydrotestosterone, or estradiol.
- Autoradiography revealed multiple alkali-resistant phosphoprotein bands (17,000–200,000 Da) in BPH tissues and neoplastic cells.
- Soluble TPK from BPH tissues exhibited a molecular weight of approximately 50,000 Da, while particulate-associated TPK eluted with proteins of Mr 210,000.
- Endogenous phosphorylation by peak fractions indicated major phosphoproteins in the 40,000–60,000 Da range.
Conclusions:
- TPKs are expressed in both benign hyperplastic prostate tissues and prostate carcinoma cell lines.
- BPH tissues exhibit at least two forms of TPK, with a predominant soluble enzyme of lower molecular weight (40,000–60,000 Da).
- The findings suggest distinct TPK profiles in benign versus malignant prostate conditions, warranting further investigation.
- TPK activity is not a reliable biomarker in seminal plasma or serum for prostate conditions.
- The characterized TPKs may represent potential targets for therapeutic intervention in prostate diseases.