Lack of interleukin 6 (IL-6) and transforming growth factor alpha (TGF-alpha) expression in chromophobe renal cell carcinomas.

We demonstrate the constitutive expression of interleukin 6 (IL-6), IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in normal kidney cells, and in the majority of renal oncocytomas, papillary and non-papillary renal cell carcinomas (RCCs) by reverse transcriptase polymerase chain reaction (RT-PCR) technique. No expression of IL-6 and TGF-alpha and variable expression of GM-CSF, IL-8, EGF and EGFR was seen in chromophobe RCCs. The lack of expression of IL-6 and TGF-alpha might be correlated with the growth pattern, poor vascularity and low malignancy of chromophobe RCCs. ImagesFigure 1

factors and their receptors. which might be involved in tumour cell proliferation via the autocrine loop. An enhanced expression of interleukin 6 (IL-6) has been associated xiith the arowth. invasixeness and chemoresistance of RCCs and w ith inflammatorv response (Miki et al. 1989: Koo et al. 1992: Gogusev et al. 1993). The constitutive expression of IL-8 and granulocyte-macrophage colonv-stimulating factor (GGM-CSF) was show n in the majoritx of RCCs (Stephens et al. 1996). The increased number of epidermal growth factor (EGF)-receptor molecules and enhanced expression of its liands EGF and transformingc,groth factor alpha (TGFa) in RCCs suggest that these genes are insolhed in the growth regulation via autocrine loop (Atlas et al. 1992: Lager et al. 1994: AoN agi et al. 1996. All these studies were camed out on RCCs in Ceneral. Recent cenetic studies. how ever. uncovered highlspecific alterations marking distinct types of renal tumours and resulted in a new classification system . The aim of this studsxwas to establish the expression profile of the abox e-mentioned genes in the four major genetic subtypes of renal cell tumours (RCe) bx the reverse transcriptase polvmerase chain reaction (RT-PCR) technique.

Tumour samples and cell culture
Fresh normal and tumour tissues w ere obtained from the Departments of Urology at the Medical School Hannover and at the Unix ersitx of Heidelberg. All tumours wxere diagnosed according to the Heidelberg Classification system . and were analysed for pathognomonic 2enetic alterations bv a microsatellite assay (Bugert et al. 1997: Palmedo et al. 1997: Schullerus et al. 1997: Herbers et al. 1998. Because tumour tissues of most RCTs contain cvtokine-producing granulocvtes. monocy-tes and macrophages. wxe anaIx-sed tumour cells gfrowinza in primarv culture or in the first passage. Cell cultures w-ere prepared by a combined enzymatic-mechanical technique described earlier ( Bugert and Koxacs. 1996). Cells w-ere rrow-n in RPMI-1640 medium (Gibco). supplemented with 10%1e fetal calf serum. No antibiotics or antimx cotics w-ere added.

RESULTS AND DISCUSSION
A panel of five normal kidney samples. six chromophobe. ten papillarn and ten non-papillar-RCCs. and ten renal oncocytomas were analysed for expression of IL-6. IL-8 and GM-CSF. as u-ell as for EGFR and its ligands. Most tumours, with exception of chromophobe RCC. showed expression of all genes analy-sed ( Table 1). The expression pattern of IL-6 and IL-8 is shown in Figure 1. As we hase analysed pure tumour cell populations. our study indicates that these cenes are expressed by the tumour cells themselves.
We have compared the expression profile and aHlelic alterations determined earlier at loci of each gene in tumour types (Bugert et al. 1997: Herbers et al. 1997: Palmedo et al. 1997: Schullerus et al. 1997. TGFax is localized on chromosome 2pI3. GM-CSF on chromosome 5q3 1. IL-6 and EGFR on chromosome 7p. and EGF and IL-8 are on chromosome 4. Our data suggest that expression of IL-6. IL-8. GM-CSF. TGFa. EGF and EGFR in RCTs. with the exception of TGFa in chromophobe RCCs. does not correlate w-ith genomic copy number in the tumour cells. Probably. these genes are expressed ubiquitouslN in all but one type of RCT. We also could not confirm the result of a recent studv showing an association between high secretion of GM-CSF and trisomy of chromosome Sq (Lahn et al. 1997).
Interleukin 6. TGFt and GM-CSF induce angiogenesis via induction of vascular endothelial growth factor (VEGF) mRNA expression (Sunderkotter et al. 1994: Cohen et al. 1996. Chromophobe RCCs are poorly s-asculanrzed and ggrow in large solid sheets of epithehal cells. This is in contrast to other types of  renal cell tumours, which are rich in vascular stroma. Although chromophobe RCCs show high mitotic activity and DNA aneuploidy, more than 90% of the patients are alive 5 years after nephrectomy indicating a low progression rate of this type of tumour (Akhtar et al, 1995;Crotty et al, 1995). It is probable that lack of, or reduced expression of, LL-6, TGFa and GM-CSF is responsible for the poor tumour angiogenesis and, consequently.
for the unique growth pattem and low malignancy of chromophobe RCCs.