Cell Lines Caki-1 Research Articles

Cloning of the human IL-13Rα1 chain and reconstitution with the IL-4Rα of a functional IL-4/IL-13 receptor complex

The human homologue of the recently cloned murine IL-13 binding protein (IL-13Rα1) was cloned from a cDNA library derived from the carcinoma cell line CAKI-1. The cloned cDNA encodes a 427 amino acid protein with two consensus patterns characteristic of the hematopoietic cytokine receptor family and a short cytoplasmic tail. The human protein is 74% identical to the murine IL-13Rα1, and 27% identical to the human IL-13Rα2. CHO cells expressing recombinant hIL-13Rα1 specifically bind IL-13 ( K d≈4 nM) but not IL-4. Co-expression of the cloned cDNA with that of IL-4Rα resulted in a receptor complex that displayed high affinity for IL-13 ( K d≈30 pM), and that allowed cross-competition of IL-13 and IL-4. Electrophoretic mobility shift assay showed that IL-13 and IL-4 were able to activate Stat6 in cells expressing both IL-4Rα and IL-13Rα1, while no activation was observed in cells expressing either one or the other alone.

Proliferation of human tumour cells after high-energy pulsed ultrasound (HEPUS) treatment

Summary. The potential anti-proliferative effect of high-energy pulsed ultrasound (HEPUS) treatment on human pancreatic (PANC-1), hepatic (SK-HEP-1) and renal (CAKI-1) tumour cells was investigated in vitro. Sonication was carried out using an experimental piezoelectric burst-signal transducer, producing bipolar oscillations with a high negative pressure amplitude. In all three cell lines tested, HEPUS-application resulted in a significant reduction (P<0.05) of vital cells. After 100 pulses 35%, 20% and 41% viable pancreatic, hepatic and renal tumour cells were detected by means of the Trypan Blue Dye exclusion test, whereas after 1000 pulses only 1.3%, 1% and 0.5% were found. A post-exposure growth delay of surviving cells, compared to control cells and cells treated with 100 pulses, was only seen in case of the CAKI-1 cell line exposed to 1000 pulses.

Estramustine-binding protein (EMBP) in renal cell carcinoma immunohistochemistry, immunoscintigraphy and in vitro estramustine effects.

The present report shows that the human renal cell carcinoma (RCC) cell lines, A498 and CAKI-2, express the estramustine-binding protein (EMBP). The RCC cell lines investigated were highly sensitive for estramustine, with cell arrest in atypical metaphase. In vitro experiments using a fluorimetric cytotoxicity assay (FMCA) showed a pronounced cytotoxic effect mediated by estramustine. Immunohistochemical analysis of tumour specimens from patients with RCC showed positive staining for EMBP in 12/16 cases. Immunoscintigraphy was performed in an experimental system in nude mice, heterotransplanted with the CAKI-2 cell line. A radiolabelled monoclonal anti-EMBP antibody was used. The results show a specific uptake of the antibody in the RCC tumour, expressed as a percentage of the injected dose per gram tissue, which ranged from 4.03 to 6.9. The results obtained form the basis for clinical studies on the feasibility of utilizing estramustine in the management of RCC. Immunoscintigraphy using the monoclonal anti-EMBP antibody is of potential use for in vivo characterization of the malignancy and in the selection patients suitable for treatment with estramustine.

Role of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) in peripheral blood mononuclear cell activation by human renal carcinoma cells.

We examined the role of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) during the activation of peripheral blood mononuclear cells (PBMCs) by the human renal carcinoma cell line CaKi-1. ICAM-1 antigen expression was induced on CaKi-1 cells by incubation with either phorbol-12-myristate 13 acetate (PMA) or interferon-gamma (IFN-gamma). Following a thorough washout of PMA and IFN-gamma and subsequent paraformaldehyde fixation, CaKi-1 cell monolayers were cocultered with allogenic PBMCs. While PMA-treated CaKi-1 cells induced PBMC proliferation and interleukin-2 receptor antigen expression, this was not the case for control or IFN-gamma-treated CaKi-1 cells. Furthermore, the induced PBMC proliferation was inhibited by specific monoclonal antibodies against ICAM-1 and LFA-1. Finally, although PMA induced human leukocyte antigen (HL)-A, B, C antigen expression on CaKi-1 cells, a monoclonal antibody against this antigen did not inhibit PBMC proliferation. We conclude that PMA can modulate CaKi-1 cells to stimulate allogenic PBMC proliferation in an ICAM-1/LFA-1 dependent, but HLA-A, B, C-independent, fashion. This stimulation might reside in the long-term activation of protein kinase C, induced by PMA.

Stimulation of intercellular adhesion molecule-1 (ICAM-1) antigen expression and shedding by interferon-gamma and phorbol ester in human renal carcinoma cell cultures: relation to peripheral blood mononuclear cell adhesion.

In the present study we investigated the effect of interferon-gamma (IFN-gamma) and phorbol-12-myristate 13 acetate (PMA) on intercellular adhesion molecule-1 (ICAM-1) antigen expression and shedding in human renal carcinoma cell cultures. We also examined the functional consequences of ICAM-1 antigen expression and soluble ICAM-1 molecules on the adhesion of peripheral blood mononuclear cells (PBMC). Incubation of the human renal carcinoma cell line CaKi-1 with IFN-gamma or PMA enhanced ICAM-1 antigen expression. The calcium ionophore, 4-bromo-calcium ionophore A23187 (Bromo-A23187) significantly enhanced the IFN-gamma and PMA effect. Soluble ICAM-1 (sICAM-1) was detected in the supernatants of stimulated but not unstimulated cultures, and correlated significantly with cellular expression. Using 51Cr-labelled peripheral blood mononuclear cells in a cell adhesion assay, we demonstrated increased adhesion in IFN-gamma-treated CaKi-1 cultures, which was augmented by Bromo-A23187. This adhesion was blocked by preincubation of CaKi-1 cells with monoclonal antibody against ICAM-1 or by preincubation of PBMC with either monoclonal antibody against leucocyte function associated antigen-1 alpha (LFA-1 alpha), a major receptor for ICAM-1, supernatants from treated cultures or purified sICAM-1 molecules. Thus, shedding of ICAM-1 may play a role during the escape from immunosurveillance by renal carcinoma cells.

Regulation of Alternative Splicing of the Fibronectin IIICS Domain by Cytokines

The primary fibronectin gene transcript is alternatively spliced in three regions, designated EIIIA, EIIIB and IIICS. While the functions of the EIIIA and EIIIB domains are still to be established, either end of the IIICS domain contains the cell-binding sites CS1 and CS5 recognised by the integrin VLA-4 which is present on mononuclear cells. We have determined the effects of three cytokines upon fibronectin mRNA and IIICS splice variants in four cultured human cell lines using Northern hybridisation and PCR. In the cell line CAKI-2, interleukin 1β, Platelet-derived growth factor BB and transforming growth factor β1 all increased mRNA splice variants where the CS1 and CS5 regions were removed. This cytokine-induced change would lead to a decrease in the CS1 and CS5 cell binding sites within the extracellular matrix. All the changes in spliceosome function increase use of downstream 3′ splice acceptor sites and may represent use of a single common pathway by three different cytokines.

Interferon-gamma increases cellular calcium ion concentration and inositol 1,4,5-trisphosphate formation in human renal carcinoma cells: relation to ICAM-1 antigen expression

In the present study, we investigated the effect of interferon-gamma (IFN-gamma) on cellular calcium ion concentration [Ca2+]i and inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) formation in the human renal carcinoma cell line CaKi-1. We also examined the possible role of a Ca(2+)-dependent mechanism during IFN-gamma-induced intercellular adhesion molecule 1 (ICAM-1) antigen expression. IFN-gamma caused a rapid concentration-dependent rise in [Ca2+]i, which was partly inhibited by diltiazem, a calcium channel blocker, TMB-8, an inhibitor of intracellular calcium redistribution, and in calcium-free medium. IFN-gamma caused a fourfold increase in Ins 1,4,5-P3 formation. The induction of ICAM-1 antigen expression was synergistically enhanced by 4-bromocalcium ionophore A23187. Finally, the calcium antagonists diltiazem. TMB-8 and EGTA, as well as two potent inhibitors of Ca(2+)-dependent kinases, calmidazolium (R24571) and W7, had no or only a minor inhibitory effect on IFN-gamma induction. Our data suggest that IFN-gamma increases [Ca2+]i in CaKi-1 cells by stimulating influx of Ca2+ and release of Ca2+ from intracellular stores, probably via Ins 1,4,5-P3 formation. IFN-gamma signal transduction in our model may not be limited to an increase in [Ca2+]i and Ins 1,4,5-P3, since IFN-gamma-induced ICAM-1 antigen expression was abrogated to a minor degree by calcium antagonists and not coupled to Ins 1,4,5-P3 formation.

Effects of pertussis and cholera toxin on the interferon-gamma stimulated immunocytochemical staining of ICAM-1 and inositol phosphate formation in a human renal carcinoma cell line.

We have recently shown that interferon-gamma (IFN-gamma) stimulated immunocytochemical staining of the intercellular adhesion molecule ICAM-1 may be dependent on inositol phosphate formation in the human renal carcinoma cell line CaKi-1. In the present study we investigated the possible role of GTP-binding proteins (G-proteins) during IFN-gamma signalling. Preincubation of CaKi-1 cells for 24 h with increasing amounts of pertussis toxin (PT) or cholera toxin (CT), two regulators of G-protein activity, inhibited IFN-gamma induced ICAM-1 staining. Preincubation with PT or CT for 24 h also inhibited IFN-gamma induced inositol 1-monophosphate (Ins 1-P), inositol 1,4 bisphosphate (Ins 1,4-P2) and inositol 1,4,5 trisphosphate (Ins 1,4,5-P3) formation. Our findings suggest that IFN-gamma induced ICAM-1 staining and inositol phosphate formation in CaKi-1 cells is dependent on a PT and CT sensitive signalling pathway. This may reflect a role for G-proteins in the coupling of IFN-gamma receptor activation and phospholipase C catalyzed phosphoinositide hydrolysis.

Comparison of the effects of tumour necrosis factor alpha stimulation and phorbol ester treatment on the immunocytochemical staining of intercellular adhesion molecule 1 in human renal carcinoma cell cultures.

Incubation of the human renal carcinoma cell line CaKi-1 with tumour necrosis factor alpha (TNF alpha) or the phorbol ester phorbol-12-myristate 13 acetate (PMA) strongly enhanced the immunocytochemical staining of the intercellular adhesion molecule ICAM-1, in a non-linear manner. Since PMA is capable of activating Ca2+/phospholipid-dependent protein kinase C (PKC), we investigated the role of this kinase during TNF alpha signal transduction. Calcium ionophore A23187 significantly enhanced PMA, but not TNF alpha-induced ICAM-1 staining. The PKC inhibitors H7, staurosporine and sphingosine abrogated the action of PMA, while TNF alpha was unaffected. Simultaneous incubation with TNF alpha and PMA resulted in maximal ICAM-1 staining significantly above values obtained when cultures were treated with either agent alone. Finally, chronic PMA treatment with subsequent TNF alpha stimulation enhanced ICAM-1 staining above values from cultures where TNF alpha was omitted. Our findings suggest that the immunocytochemical staining of ICAM-1 in CaKi-1 cells can be induced by TNF alpha through mainly PKC-independent mechanisms or by PMA through PKC-dependent mechanisms. The two agents may work synergistically in this respect.

Study of adoptive immunotherapy with lymphokine-activated killer (LAK) cells and interleukin-2 for metastatic renal cell carcinoma. I. Generation of LAK cells by incubation in serum-free medium

Investigation into the clinical application of LAK cells for treating renal cell carcinoma was carried out. LAK cells were induced from peripheral blood mononuclear cells of healthy adults or renal cell carcinoma patients by incubation of peripheral blood mononuclear cells in complete medium (RMPI1640 containing 10% heat-inactivated human AB serum) or serum-free medium (AIM-V) supplemented with IL-2. Then the characteristics of the LAK cells thus produced were studied. When cultured in complete medium, peripheral blood mononuclear cells isolated from healthy adults, recovered to 60% of the initial level on day 4 of incubation. Both NK and LAK activity were markedly enhanced before day 4. On day 4, a similar number of peripheral blood mononuclear blood cells and a similar cytotoxicity were observed in serum-free culture. The cells with a high growth rate during the 4 days of incubation were CD25, HLA-DR, CD3, CD16 cells in both cultures. The supernatant of LAK generation cultures had detectable levels of interferon (IFN)-gamma, interleukin (IL)-1 beta, and tumor necrosis factor (TNF)-alpha on day 4. IFN-gamma and IL-1 beta both showed significant concentrations in the LAK culture supernatant, which increased progressively with further culture. TNF-alpha was not produced by LAK cells alone. IFN-gamma and IL-beta production by the LAK cells was enhanced by stimulation with the Caki-1, ACHN and K-562 tumor cell lines, while TNF-alpha production was stimulated by Caki-1 and K-562 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of protein kinase C during interferon-gamma- and phorbol ester-stimulated immunocytochemical expression of ICAM-1 in human renal carcinoma cells.

Incubation of the human renal carcinoma cell line CaKi-1 with interferon-gamma (IFN-gamma) or the phorbol ester, phorbol-12-myristate 13-acetate (PMA) strongly stimulated the immunocytochemical expression of the intercellular adhesion molecule-1 (ICAM-1) in a dose-dependent manner. Since PMA is capable of activating the Ca2+/phospholipid-dependent protein kinase C (PKC), we investigated the role of this kinase during IFN-gamma signal transduction. Calcium ionophore A23187 significantly enhanced IFN-gamma- and PMA-induced ICAM-1 staining. While staurosporine, H7 and sphingosine, three known PKC inhibitors, blocked the PMA effect, only staurosporine abrogated the action of IFN-gamma. Finally, 24 h of PMA pretreatment with subsequent IFN-gamma stimulation enhanced ICAM-1 staining above values from cultures where IFN-gamma was omitted. This occurred despite the fact that 24 h of PMA pretreatment abolished the effect of IFN-gamma on PKC activation, as determined by acetylated myelin basic protein 4-14 phosphorylation. In conclusion, these results suggest that additional events other than PKC activation are required for complete regulation of ICAM-1 antigen by IFN-gamma in the whole cell population. Hence, other Ca(2+)-dependent signalling pathway(s) mediated by IFN-gamma receptors must act. Further studies are needed to elucidate these specific pathway(s) activated during IFN-gamma stimulation in our model.

The force driving the extraneuronal transport mechanism for catecholamines (uptake2).

Recently, uptake2 was shown to exist in the clonal Caki-1 cell line. The aim of this study was two-fold: a) to determine, in Caki-1 cells, the intracellular fate of 3H-noradrenaline after its translocation by uptake2 and b) to analyse the force driving uptake2. Caki-1 cells have the characteristics of a "metabolizing system" in which the activity of catechol-O-methyl transferase (COMT) greatly exceeds that of monoamine oxidase (MAO). In all subsequent experiments these enzymes were inhibited. The determination of initial rates of uptake2 into Caki-1 cells at an extracellular pH between 6.9 and 7.9 indicated that the protonated species of 3H-noradrenaline is transported. Depolarization of Caki-1 cells (by three different procedures) inhibited the inward transport. Determination of the time course of the specific accumulation of 3H-noradrenaline in Caki-1 cells and of 3H-isoprenaline in the perfused rat heart (both mediated by uptake2) revealed that depolarization (by high K+) reduced the rate constant for inward transport (kIN) and increased that for outward movement (kOUT). Consequently, depolarization reduced the steady-state factor of accumulation. It is proposed that, as the protonated species of the substrates of uptake2 is transported, the membrane potential is likely to provide the driving force for uptake2. The fact that depolarization decreased kIN and increased kOUT agrees with this proposal, as do the magnitudes of the steady-state accumulation factors determined in Caki-1 cells and perfused rat heart.

Extraneuronal noradrenaline transport (uptake2) in a human cell line (Caki-1 cells)

This study describes for the first time an experimental system for the extraneuronal transport mechanism of noradrenaline (uptake 2) which is based on a clonal cell line (Caki-1). Caki-1 cells were originally derived from a human renal cell carcinoma. The conclusion that these cells express uptake 2 is supported by several experimental findings. (1) The initial rate of 3H-noradrenaline uptake in Caki-1 cells is saturable, the Km being 450 mumol/l. (2) Inhibitors of uptake 2 such as corticosterone (1 mumol/l) and O-methyl-isoprenaline (100 mumol/l) largely inhibit 3H-noradrenaline uptake in Caki-1 cells. Whereas inhibitors of the neuronal transport mechanism for noradrenaline (uptake 1) such as desipramine (1 mumol/l) and cocaine (10 mumol/l) do not reduce it. (3) Depolarization of Caki-1 cells by the elevation of extracellular potassium inhibits 3H-noradrenaline uptake. (4) There is a highly significant correlation between the Ic50's of various compounds for the inhibition of 3H-noradrenaline uptake in Caki-1 cells and rabbit aorta known to possess uptake2. Interestingly enough, uptake 2 in Caki-1 cells and rabbit aorta is inhibited by cimetidine, quinidine and procainamide which are substrates of the renal transport mechanism for organic cations. Moreover, 3H-cimetidine is shown to be a substrate of uptake 2 in the isolated perfused rat heart. These results indicate a striking similarity between uptake 2 and the renal transport mechanism for organic cations.

Human renal carcinoma: asparagine independence with asparaginase susceptibility in culture

A human renal carcinoma cell line (Caki-1) was examined for asparagine (Asn) dependence and susceptibility to Escherichia coli asparaginase. Because this enzyme hydrolyzes glutamine (Gln) as well as Asn, even though at only 2–3% the rate, Asn − Gln + and Asn − Gln − media were prepared. Only the former supported Caki growth. The Asn − Gln − medium was then repleted with Asn, Gln, or both. Although Asn repletion failed to promote growth, addition of Gln alone or the combination supported growth as well as complete medium. With [ 3H]leucine and [ 3H]mannose incorporation to indicate protein and glycoprotein synthesis, respectively, the Gln repleted medium supported these processes as well as complete medium. Asparaginase added to complete medium was highly toxic to the Caki cells, but this is a reflection of Gln depletion rather than Asn depletion.