Matrix metalloproteinase-1 is induced by epidermal growth factor in human bladder tumour cell lines and is detectable in urine of patients with bladder tumours.

Je Nutt,J Lunec,Jk Mellon,K Qureshi

doi:10.1038/bjc.1998.467

Abstract

The matrix metalloproteinases are a family of enzymes that degrade the extracellular matrix and are considered to be important in tumour invasion and metastasis. The effect of epidermal growth factor (EGF) on matrix metalloproteinase-1 (MMP1) production in two human bladder tumour cell lines, RT112 and RT4, has been investigated. In the RT112 cell line, an increase in MMP1 mRNA levels was found after a 6-h incubation with EGF, and this further increased to 20-fold that of control levels at 24- and 48-h treatment with 50 ng ml(-1) of EGF. MMP2 mRNA levels remained constant over this time period, whereas in the RT4 cells no MMP2 transcripts were detectable, but MMP1 transcripts again increased with 24- and 48-h treatment with 50 ng ml(-1) of EGF. MMP1 protein concentration in the conditioned medium from both cell lines increased with 24- and 48-h treatment of the cells and the total MMP1 was higher in the medium than the cells, demonstrating that the bladder tumour cell lines synthesize and secrete MMP1 protein after continuous stimulation with EGF. MMP1 protein was detected in urine from patients with bladder tumours, with a significant increase in concentration with increased stage and grade of tumour. MMP1 urine concentrations may therefore be a useful prognostic indicator for bladder tumour progression.

Highlights

This induction offos mRNA shows that the bladder tumour cells responded to treatment with epidermal growth factor (EGF)
The result of probing for MMP2 transcripts is shown in Figure 1, but these remained constant with both doses of EGF for up to 4 h of treatment
Results of quantitative analysis for the MMPI transcript signals obtained on EGF stimulation of the RTI 12 cells showed a twofold increase in transcripts at 6 h when normalized with 18S levels

Summary

Methods

Two human bladder tumour cell lines, RT 112 and RT4, were obtained from ECACC. Both cell lines were positive for EGFR, confirmed by FACS analysis using the method of Brotherick et al (1994). The cells were grown routinely in medium containing 10% fetal bovine serum (FBS) and subcultured weekly. The medium was removed, and the cells were washed twice with phosphatebuffered saline (PBS) and depleted of serum by addition of medium containing 0.1% bovine serum albumin (BSA) for 24 h, apart from some controls that were maintained in 10% FBS. The cells were incubated from 30 min to 48 h, after which time medium was removed from the plates, centrifuged to remove any cells and frozen at -20°C for measurement of MMP1 concentration. The cell pellets were stored frozen at -70°C for RNA and protein extraction

Results

Discussion

Conclusion