Biomedical subjects

Johannes C Romijn

Publications and source records attributed to Johannes C Romijn.

4 recordsLinked to original sources

PubMed2005

Androgen receptor modifications in prostate cancer cells upon long-termandrogen ablation and antiandrogen treatment.

To study the mechanisms whereby androgen-dependent tumors relapse in patients undergoing androgen blockade, we developed a novel progression model for prostate cancer. The PC346C cell line, established from a transurethral resection of a primary tumor, expresses wild-type (wt) androgen receptor (AR) and secretes prostate-specific antigen (PSA). Optimal proliferation of PC346C requires androgens and is inhibited by the antiandrogen hydroxyflutamide. Orthotopic injection in the dorsal-lateral prostate of castrated athymic nude mice did not produce tumors, whereas fast tumor growth occurred in sham-operated males. Three androgen-independent sublines were derived from PC346C upon long-term in vitro androgen deprivation: PC346DCC, PC346Flu1 and PC346Flu2. PC346DCC exhibited androgen-insensitive growth, which was not inhibited by flutamide. AR and PSA were detected at very low levels, coinciding with background AR activity in a reporter assay, which suggests that these cells have bypassed the AR pathway. PC346Flu1 and PC346Flu2 were derived by culture in steroid-stripped medium supplemented with hydroxyflutamide. PC346Flu1 strongly upregulated AR expression and showed 10-fold higher AR activation than the parental PC346C. PC346Flu1 proliferation was inhibited in vitro by R1881 at 0.1 nM concentration, consistent with a slower tumor growth rate in intact males than in castrated mice. PC346Flu2 carries the well-known T877A AR mutation, causing the receptor to become activated by diverse nonandrogenic ligands including hydroxyflutamide. Array-based comparative genomic hybridization revealed little change between the various PC346 lines. The common alterations include gain of chromosomes 1, 7 and 8q and loss of 13q, which are frequently found in prostate cancer. In conclusion, by in vitro hormone manipulations of a unique androgen-dependent cell line expressing wtAR, we successfully reproduced common AR modifications observed in hormone-refractory prostate cancer: downregulation, overexpression and mutation.

Androgen Antagonists↗

PubMed2003

Internalization of calcium oxalate crystals by renal tubular cells: a nephron segment-specific process?

BACKGROUND: Crystal retention in the kidney is caused by the interaction between crystals and the cells lining the renal tubules. These interactions involve crystal attachment, followed by internalization or not. Here, we studied the ability of various renal tubular cell lines to internalize calcium oxalate monohydrate (COM) crystals. METHODS: Crystal-cell interactions are studied by light-, electron-, and confocal microscopy with cells resembling the renal proximal tubule [porcine kidney (LLC-PK1)], proximal/distal tubule [Madin-Darby canine kidney II (MDCK-II)], and distal tubule and/or collecting ducts [(Madin-Darby canine kidney I (MDCK-I), rat cortical collecting duct 1 (RCCD1)]. Crystal-binding strength and internalization are characterized and quantified with radiolabeled COM. RESULTS: Microscopy studies showed that crystals were firmly embedded in the membranes of LLC-PK1 and MDCK-II cells to be subsequently internalized. On the other hand, crystals bound only loosely to MDCK-I and RCCD1 and were not taken up by these cells. Crystal uptake by LLC-PK1 and MDCK-II, expressed in microg/10(6) cells, is temperature-dependent and gradually increases from 0.88 and 0.15 in 30 minutes, respectively, to 4.70 and 3.85, respectively, after five hours, whereas these values never exceeded background levels in MDCK-I and RCCD1 cells. CONCLUSION: The adherence of COM crystals to renal cells with properties of the proximal tubule is inevitable and actively followed by their uptake, whereas crystals attached to cells resembling the distal tubule and/or collecting duct are not internalized. Since crystal formation usually occurs in segments beyond the renal proximal tubule, crystal uptake may be of less importance in the etiology of idiopathic calcium oxalate stone disease.

Animals↗

PubMed2003

Pericellular matrix formation by renal tubule epithelial cells in relation to crystal binding.

BACKGROUND/AIM: Retention of crystals in the kidney ultimately leads to renal stone formation. Hyaluronan (HA) has been identified as binding molecule for calcium oxalate monohydrate crystals. The association of high molecular mass (M(r)) HA with cell surface receptors such as CD44 gives rise to pericellular matrix (PCM) formation by many eukaryotic cells in culture. Here, we study the ability of several renal tubular cell lines to assemble PCMs and to synthesize high-M(r) HA during proliferation in relation to crystal retention. METHODS: PCM assembly by MDCK-I, MDCK-II, and LLC-PK1 cells was visualized by particle exclusion assay. Metabolic labeling studies were performed to estimate the cellular production of HA. The expression of CD44 and HA was studied using fluorescent probes, and crystal binding was quantified with radiolabeled calcium oxalate monohydrate. RESULTS: PCMs were formed, and HA was expressed by most MDCK-I and some MDCK-II, but not by LLC-PK1 cells. All cell types expressed CD44 at their apical surface. MDCK-I and MDCK-II cells secreted, respectively, 14.7 +/- 1.6 and 0.5 +/- 0.2 pmol [3H]glucosamine incorporated in high-M(r) HA, whereas LLC-PK1 cells did not secrete HA. Streptomyces hyaluronidase treatment significantly decreased crystal binding (microg/cm2) to MDCK-I cells (from 8.6 +/- 0.4 to 3.9 +/- 0.9), but hardly to MDCK-II cells (from 10.2 +/- 0.2 to 9.6 +/- 0.1) or LLC-PK1 cells (from 10.2 +/- 0.8 to 9.9 +/- 0.3). CONCLUSIONS: There are various forms of crystal binding to renal tubular cells in culture. Crystal attachment to MDCK-I and some MDCK-II cells involves PCM assembly that requires high-M(r) HA synthesis. HA production and PCM formation do not play a role in crystal binding to LLC-PK1 and the majority of MDCK-II cells. It remains to be determined which form of binding is involved in renal stone disease.

Animals↗

PubMed2002

Urinary crystallization inhibitors do not prevent crystal binding.

PURPOSE: Renal stone formation requires the persistent retention of crystals in the kidney. Calcium oxalate monohydrate (COM) crystal binding to Madin Darby canine kidney strain I (MDCK-I), a cell line that resembles the epithelium in the renal distal tubule/collecting duct, is developmentally regulated, while LLC-PK1 cells (American Type Tissue Collection), which are widely used as a model of the renal proximal tubule, bind crystals irrespective of their stage of epithelial development. Whereas to our knowledge the binding molecules for COM at the surface of LLC-PK1 cells are still unknown, crystals adhere to the hyaluronan (HA) rich pericellular matrix transiently expressed by mobile MDCK-I cells. In the current study we investigated whether crystal binding to either cell type is influenced by urinary substances, including glycoprotein inhibitors of crystallization MATERIALS AND METHODS: We studied crystal binding to MDCK-I cells during wound repair, to confluent LLC-PK1 cells and to HA immobilized on a solid surface using [14C] COM pretreated or not pretreated with urine from healthy male volunteers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were performed to assess whether the crystals became coated with urine derived proteins RESULTS: Western blot analysis demonstrated that pretreated COM crystals were covered with protein inhibitors of crystallization. However, this protein coat had no significant effect on the level of crystal binding to either cell type. In contrast, the adherence of urine treated crystals to immobilized HA was significantly reduced CONCLUSIONS: The adherence of crystals to pericellular matrixes may encompass more than their simple fixation to the polysaccharide HA. Calcium oxalate crystal retention is not prevented by coating crystals with urinary constituents such as glycoproteins and, therefore, may predominantly depend on the surface properties of the renal tubular epithelium.

Animals↗