Restoration of contact inhibition in human glioblastoma cell lines after MIF knockdown.

BACKGROUND: Studies of the role of the cytokine macrophage-migration-inhibitory-factor (MIF) in malignant tumors have revealed its stimulating influence on cell-cycle progression, angiogenesis and anti-apoptosis. RESULTS: Here we show that in vitro targeting MIF in cultures of human malignant glioblastoma cells by either antisense plasmid introduction or anti-MIF antibody treatment reduced the growth rates of tumor cells. Of note is the marked decrease of proliferation under confluent and over-confluent conditions, implying a role of MIF in overcoming contact inhibition. Several proteins involved in contact inhibition including p27, p21, p53 and CEBPalpha are upregulated in the MIF antisense clones indicating a restoration of contact inhibition in the tumor cells. Correspondingly, we observed a marked increase in MIF mRNA and protein content under higher cell densities in LN18 cells. Furthermore, we showed the relevance of the enzymatic active site of MIF for the proliferation of glioblastoma cells by using the MIF-tautomerase inhibitor ISO-1. CONCLUSION: Our study adds another puzzle stone to the role of MIF in tumor growth and progression by showing the importance of MIF for overcoming contact inhibition.

Differential expression of somatostatin receptor subtypes in hepatocellular carcinomas.

BACKGROUND/AIMS: Somatostatin analogues inhibit cell proliferation by stimulation of distinct somatostatin receptor (SSTR) subtypes. In recent years, these compounds have been introduced into the therapy of advanced hepatocellular carcinoma (HCC). The efficacy of this treatment is under debate due to the controversial results of clinical trials. Despite the widespread clinical use of somatostatin analogues in HCC, little is known about the expression of each of the five SSTRs in these tumors. METHODS: We analyzed the expression of SSTR subtypes in 56 HCCs by immunohistochemistry using subtype-specific antibodies. Six of the samples were also investigated by RT-PCR using subtype-specific oligonucleotide primers. RESULTS: HCCs display differential, individual expression patterns as well as variable expression levels for SSTRs. The overall expression rate of SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 is 46, 41, 64, 0, and 75%, respectively. No significant correlation was observed between SSTR expression and tumor stage, differentiation, histological tumor type, or underlying liver disease. CONCLUSIONS: Individual patterns and levels of SSTR expression might determine the response to treatment with somatostatin analogues in HCC. Selective treatment of these tumors based on the analysis of SSTR subtype expression might lead to an increase in response rates.

The cholecystokinin2-receptor mediates calcitonin secretion, gene expression, and proliferation in the human medullary thyroid carcinoma cell line, TT.

Gastrin-induced release of calcitonin from medullary thyroid carcinomas (MTC) is based on the expression of the cholecystokinin(2)-receptor (CCK(2)R) in these tumors. Recently, we have shown that the CCK(2)R is expressed not only in MTC but also in C-cells within the normal thyroid gland. The functions of the CCK(2)R in MTC and C-cells are largely unknown. We therefore explored the effects of gastrin-induced CCK(2)R stimulation in the highly differentiated MTC cell line, TT. CCK(2)R expression in TT-cells is detectable by RT-PCR as well as immunocytochemistry. Stimulation of the CCK(2)R by gastrin induces immediate release of calcitonin from TT-cells. Moreover, quantitative (LightCycler) RT-PCR demonstrates that gastrin stimulates transcription of the calcitonin and chromogranin A genes in TT-cells. TT-cell proliferation, assessed by counting of viable cells and (3)H-thymidine uptake, is markedly increased by gastrin. This effect is inhibited by the CCK(2)R-specific antagonist L-365,260. Our findings suggest physiological functions for the CCK(2)R in calcitonin-secretion and gene expression as well as a pathophysiological role in MTC proliferation. CCK(2)R antagonists might have therapeutic potential in these tumors.

Expression of the cholecystokinin 2-receptor in normal human thyroid gland and medullary thyroid carcinoma.

OBJECTIVE: The cholecystokinin(2)-receptor (CCK(2)R) promotes secretion and cell growth induced by its ligands cholecystokinin (CCK) and gastrin. The receptor has recently been shown to be expressed in human medullary thyroid carcinomas (MTCs). The objective of this study was to analyze CCK(2)R expression in MTC samples of different tumor stages as well as in non-malignant thyroid tissues. DESIGN AND METHODS: Using RT-PCR we investigated 19 MTC samples and TT-cells (a human MTC cell line), as well as samples of normal thyroid. In addition, we performed immunohistochemistry using calcitonin- and CCK(2)R-specific antibodies on MTCs and samples of C-cell hyperplasia. RESULTS: We demonstrate for the first time that CCK(2)R is expressed not only in MTCs but in all samples of normal thyroid tissue. Using immunohistochemistry the receptor could be localized on calcitonin-secreting C-cells. The highest incidence of CCK(2)R expression in MTCs was observed in early-tumor stages, whereas CCK(2)R could not be detected in advanced or metastasized tumors. CONCLUSIONS: The expression of CCK(2)R in C-cells suggests a physiological function for gastrin and/or CCK in the regulation of calcitonin release, presumably related to bone and calcium metabolism. Moreover, these ligands might act as growth factors in MTCs. Efforts in the development of CCK(2)R scintigraphy for the detection of MTC lesions might have to consider a lower incidence of the receptor in advanced tumor stages.

Gastrin releasing peptide-preferring bombesin receptors mediate growth of human renal cell carcinoma.

Bombesin-like peptides typically act as neurotransmitters along the brain-gut axis and as growth factors in various human tissues. The present study demonstrates the expression of gastrin releasing peptide (GRP)-preferring bombesin receptors in human renal cell carcinoma but not in normal kidney tissue. The expression of GRP receptors was characterized at the mRNA level by reverse transcription-PCR, as well as at the protein level by binding of (125)I-[Tyr(4)] bombesin to membranes prepared from tumor tissue (K(d) 0.3 nM) and healthy kidney tissue from the same four patients. GRP receptors were also demonstrated in four human kidney carcinoma cell lines (A-498, CAKI-1, CAKI-2, and ACHN). The effects of bombesin/GRP agonists and/or antagonists on growth were investigated in vitro on CAKI-2 cells, which expressed large amounts of GRP receptors. Cell numbers stimulated by 10% fetal calf serum were significantly stimulated by interleukin-1beta (control) and GRP-7 (10(-7) M), both in the range of 136 to 148%; addition of the GRP receptor antagonist acetyl-GRP(20-27) (10(-6) M) completely reversed this effect. Bombesin alone (10(-6) M) significantly stimulated CAKI-2 cells (129%) cultured with 0.5% fetal calf serum, whereas another antagonist, D-Phe6,Leu13,(CH2NH)Leu14 bombesin(6-14) (1 microM), alone did not inhibit growth, thus excluding an autocrine mechanism. These results indicate for the first time that malignant transformation of human kidney tissue into renal cell carcinoma is accompanied by novel expression of GRP receptors. Bombesin-like peptides might act as mitogens in these carcinomas, and they might be useful as diagnostic or therapeutic tools such as tumor imaging or internal radiotherapy.