Efectos generales de la descentralización 1 Resultados de estudios cuantitativos

2.6.1 Expression pattern of VHL mRNA

Latif et al. identified the VHL gene and

showed Northern blot analysis of VHL gene expression in foetal and adult kidney and brain as well as in adult adrenal and prostate [81]. However the first comprehensive study addressing expression of the von Hippel-Lindau tumour suppressor gene was undertaken by Kessler et al. and published in 1995 [94]. Here the author’s investigated expression of the VHL gene in human foetal kidney, and during mouse embryogenesis using in situ hybridisation with 35_{S-labelled anti-sense VHL probes}

derived from human and mouse cDNAs on cryosections of human foetal kidney and paraffin sections of murine embryos. The results concluded that in human foetal kidney, the enhanced epithelial expression of the VHL gene is consistent with the role of this gene in renal cell carcinoma. Furthermore, the report showed widespread expression of the VHL gene during embryogenesis. An interesting observation apparent from the in situ investigation was the differential developmental VHL expression within epithelial cells derived from mesoderm (kidney and epididymis), endoderm (lung and pancreas), and ectoderm (eye). In addition, the localisation of VHL expression in human and mouse nephrogenesis was consistent with the histo-pathologic studies of the origin of the clear cell phenotype of renal cell carcinoma and their associated VHL mutations [95]. To complement this work,

Table 7. Summary of VHL mRNA expression during human embryogenesis Germ Layer Tissue VHL Expression Endoderm Gut Pancreas Lung Pharyngeal pouches - - ++ - Ectoderm

Dorsal root ganglia Periderm

Brain

Cranial ganglia (V, VII, VIII) Vestibular apparatus Eye Cephalic mesenchyme + (+) ++ +++ +++ + + Mesoderm Heart Indifferent gonad Mesonephric duct Paramesonephric duct Bowman’s capsule Loop of Henle Nephrogenic cord

Metanephric collecting ducts Proximal convoluted tubule Perichondrium

Liver (haematopoietic tissue) Adrenal Striated muscle (+) ++++ ++ ++ + ++++ + ++ ++ (+) + (+) +

Figure 20. Quantitative RT-PCR of GAPDH and VHL mRNA in 8-10 week human foetal tissues. VHL(1) = wt-VHL; VHL(2) = ∆Exon2 VHL; Li=liver; Ki=kidney; Br=brain; Ey=eye; SC=spinal cord; Ad=adrenal; In=intestine; Pa=pancreas; He=heart; Te=testis; Lu=lung; Lm=limbs; Pl=placenta; Tu=adult proximal renal tubule cell culture.

Adapted from Richards et al.1996 Adapted from Richards et al.1996

Richards et al. investigated the expression of VHL mRNA during human embryogenesis

again by in situ hybridisation studies at 4, 6 and 10 weeks post conception. Although

VHL mRNA was expressed in all three germ layers, they noted strong expression in the

central nervous system, kidneys, testis and lung. Within the kidney, VHL mRNA was differentially expressed within renal tubules, and the authors reiterated the previous findings by suggesting that the VHL gene product may have a specific role in kidney development.

Moreover, two alternatively spliced VHL mRNAs characterised by inclusion (isoform I) or exclusion (isoform II) of exon 2 were transcribed in adult tissues. To investigate if the two isoforms were differentially expressed during embryogenesis, VHL mRNA was reverse transcribed from 13 foetal tissues (8-10 weeks gestation). The quantitative distribution of VHL mRNA within foetal tissues reflected that seen by in situ hybridisation and the ratio of the two VHL isoforms was similar between tissues (Fig.20).

2.6.2 Expression pattern of pVHL

Los et al. [96] first addressed expression of VHL protein in human tissues in 1996. The

VHL protein was widely expressed in normal human tissue. The authors recorded a cellular distribution of the protein that was confined to the cytoplasm of specific cell types. Immuno-histochemistry did not facilitate the discrimination of tumours obtained from VHL patients or tumours unrelated to the VHL disease. Renal cell carcinomas, haemangioblastomas, and phaeochromocytomas, either VHL-related or sporadic, demonstrated positive staining for the VHL protein, which suggests that the antibody used recognised mutated VHL protein. However, no consideration was given to alternative pVHL species, e.g. pVHL19. The antibodies used were unexplained, the

epitopes not described, and no proof of specificity in terms of western blot analysis or immuno-precipitation of endogenous pVHL was demonstrated. Without such pertinent data, drawing conclusions is difficult.

A year later, Corless et al. re-addressed the issue of pVHL expression in normal and neoplastic tissues [3]. At this time, epitope-tagged pVHL had been observed in either the nucleus or the cytoplasm of cultured cells, depending on the density of the cell culture [97-99]. In this article, the cellular localisation of pVHL in normal and neoplastic human tissues was documented using three different monoclonal antibodies, one being a commercially available antibody widely used and characterised, Ig32. This monoclonal antibody recognises both pVHL30 and pVHL19. The other two antibodies had epitopes

mapped to a region of amino acids 72-120, hence total pVHL was being detected. Strong expression of pVHL was observed in the epithelial cells of all organs examined, particularly in renal tubules, and was exclusively cytoplasmic. Lesser degrees of staining, also cytoplasmic, were observed in other cell types.

A variety of carcinomas (lung, prostate, colon, breast, bladder, and thyroid) showed strong cytoplasmic staining for pVHL including four of five sporadic clear cell RCC. Of the non-epithelial neoplasms examined, only one tumour, an embryonal rhabdomyosarcoma18, failed to stain for pVHL. The findings established widespread expression of VHL at the protein level and the authors state that it provided strong evidence that most, if not all, pVHL is localised to the cytoplasm of cells in vivo. We now know that this is in fact not the case, and pVHL can be both nuclear and cytoplasmic (see results).

From these studies we can conclude that the areas of highest expression do not completely correlate with the tissues that are involved in VHL disease. The tissue specificity of VHL disease therefore cannot be entirely explained by tissue-specific expression during either foetal development or adulthood. However, the antibodies used would not distinguish between the different sized pVHL’s generated by alternative translation initiation sites. It is interesting to note that subsequent studies like those mentioned have documented VHL mRNA in a wide variety of tissues in a pattern similar to the generalised expression of other tumour suppressor genes including retinoblastoma and p53 [100, 101].

Table 8. Summary of normal human tissues positive for pVHL immunostaining

Epithelial Cells Muscle Cells Lymphoid Cells Other

Renal tubule Renal glomerulus Colon Ileum Endometrium Bladder Prostate Thyroid follicle Bile duct

Uterine smooth muscle Intestinal smooth muscle Vascular smooth muscle

Skeletal muscle Lymphocytes Endothelial cells Sertoli cells Leydig cells Spermatogonia / spermatocytes Peripheral nerve and

ganglion cells Adrenal cortical cells

Table 9. Summary of human tumours positive for pVHL immunostaining

Renal Tumours Other Tumours Other Carcinomas Germ Cell Tumours

Clear cell carcinoma Papillary carcinoma Wilms’ tumour Phaeochromocytoma Malignant mesothelioma (pleural)

Islet cell tumour Epithelioid

haemangioendothelioma Leiomyoma, uterus Sex cord stromal tumour (ovary)

Thymoma

Squamous cell carcinoma (lung, oral cavity)

Small cell carcinoma (lung) Adenocarcinoma (lung) Adenocarcinoma (prostate) Transitional cell carcinoma (bladder)

Cholangiocarcinoma (liver) Ductal carcinoma (breast) Papillary thyroid carcinoma

Seminoma (testis) Teratoma (testis) Embryonal carcinoma (testis)

Adaped from Corless et al. 1997 Adaped from Corless et al. 1997