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Biomedical subjects

R K Puri

Publications and source records attributed to R K Puri.

At least 73 records · Page 4Linked to original sources
PubMed1996

Targeting of interleukin-13 receptor on human renal cell carcinoma cells by a recombinant chimeric protein composed of interleukin-13 and a truncated form of Pseudomonas exotoxin A (PE38QQR).

We have previously shown that human renal cell carcinoma (RCC) cells express large numbers of interleukin-13 receptors (IL-13R), a newly described hemopoietic growth factor receptor. To target tumor cells that express IL-13R, we have produced a chimeric protein composed of human IL-13 and a derivative of Pseudomonas exotoxin A, termed PE38QQR. We report here that IL13-PE38QQR is highly cytotoxic to many human RCC cell lines. IL-13R-negative cell lines or cell lines expressing low numbers of IL-13R ( < 300 sites/cell) that include human bone marrow-derived cells were not susceptible to the cytotoxic effect of IL 13-PE38QQR. The sensitivity of RCC cells to IL13-PE38QQR correlated positively with the density of IL-13R. The cytotoxic activity of IL13-PE38QQR was competed by an excess of IL-13 in a protein synthesis inhibition assay and confirmed by a clonogenic assay. Even though IL-13 and IL-4 are homologues and IL-4R and IL-13R have been proposed to share a receptor subunit, IL-4 did not compete for the cytotoxicity mediated by IL13-toxin on RCC. IL13-PE38QQR competes for [125I]-IL-13 binding sites on RCC cells, although at a lower affinity than the wild-type recombinant cytokine. Human T-cell, B-cell, and monocytic cell lines are unresponsive to the cytotoxic action of IL13-PE38QQR. Thus, our results indicate that IL13-PE38QQR is highly cytotoxic to human RCC cells, although it is not cytotoxic to a variety of normal hematopoietic cells. IL13-PE38QQR should be further investigated preclinically for the treatment of human RCCs.

ADP Ribose Transferases↗
PubMed1996

IL-13 induces phosphorylation and activation of JAK2 Janus kinase in human colon carcinoma cell lines: similarities between IL-4 and IL-13 signaling.

We have recently reported that IL-13R may share a component with IL-4R. Here we report that both IL-4 and IL-13 share signaling events in human colon carcinoma cell lines (HT-29 and WiDr). IL-13 caused rapid phosphorylation of the three out of four members of the known Janus family of kinases (JAKs). We show that JAK2 kinase is rapidly phosphorylated and activated in response to IL-13. Within 1 min of activation, JAK2 was phosphorylated, and peaked in 10 min. In addition, IL-13 phosphorylated insulin response substrate-1, IL-4R p140, JAK1, and Tyk2, but not JAK3 kinase. IL-4 also stimulated all three kinases and substrates, but unlike in immune cells, IL-4 did not involve JAK3 activation for its signaling in colon cancer cell lines. Furthermore, JAK2 associated with the IL-4R p140 before and after stimulation with IL-13. Both IL-13 and IL-4 induced phosphorylation of IL-4 STAT (STAT6) but not STAT1, STAT3, or STAT5. 125I-IL-13 did not bind to colon cancer cell lines, but unlabeled IL-13 competed for the binding of 125I-IL-4. Our data suggest that IL-13 utilizes IL-4R and its signaling pathway, and JAK2 may play an important role in the function of IL-4R and IL-13R in colon cancer cells.

Antigens, CD↗
PubMed1996

Ehretianone, a novel quinonoid xanthene from Ehretia buxifolia with antisnake venom activity.

Ehretianone (1), a new quinonoid xanthene, together with known sterols, was isolated from a MeOH extract of the root bark of Ehretia buxifolia. The structure of ehretianone was elucidated as 7-hydroxy-9a alpha-(3-methylbut-2-enyl)-4a alpha,9 alpha-(2-methylprop-2-enyl)-4a, 9a-dihydro-1,4-dioxoxanthene on the basis of spectroscopic data and X-ray crystallographic analysis. The antisnake venom activity of ehretianone against Echis carinatus venom in mice is also reported.

Animals↗
PubMed1996

Interleukin 13 inhibits growth of human renal cell carcinoma cells independently of the p140 interleukin 4 receptor chain.

Interleukin-13 (IL-13) is a cytokine produced primarily by activated T lymphocytes. It exerts a variety of effects on different cell types, including monocytes, B lymphocytes, mast cells, and keratinocytes. The effects of IL-13 on target cells are often similar to the effects of IL-4, which is another cytokine product of activated T lymphocytes. We recently described the expression of intermediate- to high-affinity receptors for IL-13 (IL-13R) on renal cell carcinoma (RCC) cells. In the present study, we examined the effect of IL-13 on the growth of RCC cells as measured by [3H]thymidine uptake and a clonogenic assay. In addition, we used an IL-4R-specific antibody to examine the specificity of IL-4R and IL-13R binding and function. We observed that IL-13 inhibited RCC cell proliferation by up to 50% and colony formation by up to 32% when compared with cells cultured in medium alone. A combination of IL-4 and IL-13 did not have an additive or synergistic effect on the growth of RCC cells. These cells expressed mRNA for IL-13 and secreted immunoreactive IL-13 protein in culture. The growth-inhibitory effects of IL-13 were specific, because they were not affected by antibodies to IL-4 or to the 140-kilodalton subunit of IL-4R. Furthermore, polyclonal antibodies to IL-4R failed to inhibit the binding of 125I-IL-13 to RCC cells. These results indicate that IL-13 has significant antiproliferative effects on human RCC cells, and the inhibition of IL-13 effects by anti-IL-4R antibody previously reported in lymphoid cells does not occur in RCC cells.

Antibodies↗
PubMed1995

Receptors for interleukin (IL)-4 do not associate with the common gamma chain, and IL-4 induces the phosphorylation of JAK2 tyrosine kinase in human colon carcinoma cells.

We have previously reported on the expression of interleukin-4 receptors (IL-4R) on many human epithelial cancer cells; however, the binding characteristics, structure, function, and signal transduction through the IL-4R in cancer cells is not known. IL-4 binding characteristics were determined in human colon carcinoma cell lines by a 125I-IL-4 binding assay, which demonstrated that the HT-29 and WiDr colon cancer cell lines expressed high affinity IL-4R (Kd = 200 pM). Cross-linking experiments revealed a major band of 140 kDa and a broad band at 70 kDa. While the common gamma chain of IL-2R is associated with IL-4R in immune cells and is similar in size to the 70-kDa protein, this chain was not expressed in these colon cancer cells. Interestingly, IL-13, which has many functions similar to IL-4, inhibited 125I-IL-4 binding to both the 140- and 70-kDa molecules. Next, we investigated the mechanism of IL-4-induced signal transduction in colon cancer cells. After stimulation with IL-4, a 170-kDa band was primarily phosphorylated within 1 min of exposure and was identified as insulin receptor substrate-1. In addition, by immunoprecipitation assay, three other phosphorylated bands were identified as JAK1, JAK2, and Tyk2 tyrosine kinases. The phosphorylation of JAK1 and JAK2 was induced by IL-4 stimulation; however, Tyk2 was constitutively phosphorylated, and IL-4 treatment further augmented this phosphorylation. The kinetics and in vitro kinase assays demonstrated that JAK1, JAK2, and Tyk2 were phosphorylated within minutes and that JAK1 and JAK2 were activated after IL-4 exposure. Contrary to observations in immune cells. JAK3 mRNA was neither detected in colon cancer cells nor did IL-4 treatment cause phosphorylation of JAK3. These data indicate that in colon carcinoma cells JAK1, JAK2, Tyk2, and insulin receptor substrate-1 are phosphorylated after IL-4 stimulation. In addition, as is the case in lymphoid cells, IL-4 activated and phosphorylated signal transducers and activators of transcription (IL-4-STAT or STAT-6) protein in both colon cancer cell lines. These results indicate that the IL-4R complex is composed of different subunits in different tissues and shares a component with the IL-13R complex. In addition, we demonstrate for the first time that like its family members (e.g. IL-3 and GM-CSF), IL-4 can phosphorylate and activate JAK-2 kinase.

Antigens, CD↗
PubMed1995

Increased antitumor activity of a circularly permuted interleukin 4-toxin in mice with interleukin 4 receptor-bearing human carcinoma.

We reported previously that circularly permuted interleukin-4 (IL4), composed of amino acids 38-129 of IL4 connected by a linker peptide GGNGG to amino acids 1-37, is preferable to native IL4 for fusing to the amino terminus of truncated Pseudomonas exotoxin (PE) to make a recombinant toxin, because the new ligand-toxin junction results in improved IL4 receptor (IL4R)-binding (R. J. Kreitman et al., Proc. Natl. Acad. Sci. USA, 91: 6889-6893, 1994). We now report that the improved binding of circularly permuted IL4-toxin is associated with improved antitumor activity in tumor-bearing mice. For in vivo testing, we made an improved circularly permuted IL4-toxin, termed IL4(38-37)-PE38KDEL. It contains an N38D mutation at the amino terminus, allowing improved expression and large-scale production in Escherichia coli. It also contains the truncated toxin PE38KDEL, which is composed of amino acids 253-364 and 381-608 of PE, followed by KDEL. To evaluate antitumor activity, nude mice carrying s.c. tumors composed of IL4R-bearing human A431 epidermoid carcinoma cells were injected with recombinant toxins i.v. every other day for three doses. IL4(38-37)-PE38KDEL induced complete remissions in 80% of mice receiving 50 micrograms/kg x 3 and 100% of mice receiving 100 micrograms/kg x 3, while only 70% of mice receiving 200 micrograms/kg x 3 of the native IL4-toxin IL4-PE38KDEL obtained complete remission. Disease-free survival after obtaining complete remissions was higher in mice treated with IL4(38-37)-PE38KDEL 50 micrograms/Kg QOD x 3 than with IL4-PE38KDEL 200 micrograms/Kg QOD x 3 (P < 0.03). IL4(38-37)-PE38KDEL and IL4-PE38KDEL exhibited similar toxicity and pharmacokinetics in the mice, indicating that the improved antitumor activity of the circularly permuted IL4-toxin was due to its improved binding to the IL4R on the target cells.

ADP Ribose Transferases↗
PubMed1995

A novel chimeric protein composed of interleukin 13 and Pseudomonas exotoxin is highly cytotoxic to human carcinoma cells expressing receptors for interleukin 13 and interleukin 4.

Chimeric proteins provide a unique opportunity to target therapeutic bacterial toxins to a subset of specific cells. We have generated a new recombinant chimeric toxin composed of human interleukin 13 (hIL13) and a Pseudomonas exotoxin A (PE) mutant, PE38QQR. The hIL13-PE38QQR chimera is highly cytotoxic to cell lines derived from several human epithelial carcinomas such as adenocarcinoma of stomach, colon, and skin. The cytotoxic action of hIL13-PE38QQR, which can only occur upon internalization of ligand-receptor complex, is blocked by an excess of hIL13 but not of hIL2. This action is not solely hIL13-specific because an excess of hIL4 also blocks the cytotoxicity of hIL13-toxin. Conversely, hIL13 blocks the cytotoxicity of a hIL4-PE38QQR chimera. Binding studies showed that hIL13 displaces competitively 125I-labeled hIL4-PE38QQR on carcinoma cells. These results indicate that IL4 and IL13 compete for a common binding site on the studied human cell lines. Despite this competition, hIL4 but not hIL13 decreased protein synthesis in malignant cells susceptible to the cytotoxicity of both hIL13- and hIL4-PE38QQR. Our results suggest that a spectrum of human carcinomas express binding sites for IL13. Furthermore, hIL13 and hIL4 compete reciprocally for a form of the receptor that is internalized upon binding a ligand. Thus, cancer cells represent an interesting model for studying receptors for these two growth factors. Finally, hIL13-PE38QQR may be a useful agent in the treatment of several malignancies.

ADP Ribose Transferases↗
PubMed1995

Up-regulation of intercellular adhesion molecule 1 (ICAM-1) on human renal cell carcinoma cells by interleukin-4.

We have previously demonstrated that human renal cell carcinoma (RCC) cells express high-affinity IL-4 receptors (IL-4R). To study the functions of these receptors, we have examined the effect of IL-4 on the expression of intercellular adhesion molecule-1 (ICAM-1 or CD54) on human RCC cells. Following incubation with various concentrations of IL-4, RCC cells were examined for ICAM-1 expression by flow cytometric analysis. The 2 primary RCC cell cultures and the 2 cell lines examined expressed varying basal levels of ICAM-1 on the cell surface. IL-4 treatment increased ICAM-1 expression in a time-dependent manner and maximum augmentation of ICAM-1 expression was observed after a 48 hr incubation. The increase in ICAM-1 expression was specific because anti-hIL-4 antibody blocked this effect. No enhancement of ICAM-1 expression was observed when RCC cells were incubated with IL-4 in the presence of cycloheximide, indicating that the IL-4 effect requires new protein synthesis. Up-regulation of ICAM-1 expression was also observed at the mRNA level and maximum increase in message occurred 8 hr post-IL-4 treatment. Both IL-4 and IFN-gamma also increased soluble ICAM-1 levels in WS-RCC culture supernatant. The significance of enhanced soluble and surface ICAM-1 expression was investigated by examining the lymphokine activated killer (LAK) cell-mediated lysis of IL-4-treated WS-RCC cells. LAK cells lysed WS-RCC cells very effectively, but lysis observed in target cells pre-treated with IL-4 did not correlate with the increased expression of ICAM-1 antigen. Our results indicate a previously unknown function of IL-4 on RCC and further demonstrate that IL-4R on RCC are functional.

Blotting, Northern↗
PubMed1995

Receptor for interleukin 13. Interaction with interleukin 4 by a mechanism that does not involve the common gamma chain shared by receptors for interleukins 2, 4, 7, 9, and 15.

Interleukin 13 (IL-13) shares many biological properties with IL-4, and although the receptor for IL-4 (IL-4R) has been characterized, the expression and structure of IL-13 receptor are unknown. We report here that human renal cell carcinoma (RCC) cells express large numbers of functional IL-13R. Human B lymphocytes and monocytes expressed a very small number of IL-13R, while resting or activated human T cells expressed little or no IL-13R. IL-4 did not compete for IL-13 binding, while IL-13 competed for IL-4 binding, even though IL-4R and IL-13R are structurally distinct on human RCC cells. IL-13 cross-linked with one major protein that is similar in size to the gamma c subunit of IL-2, -4, -7, -9, and -15 receptors but was not recognized by anti-gamma c or anti-IL-4R antibodies. IL-4, on the other hand, cross-linked with two major proteins, the smaller of which appears to be similar in size to IL-13R and gamma c, but (like the IL-13R) it did not react with anti-gamma c antibody. Although as shown in this study and in previous studies, gamma c is a functional component of IL-4R in lymphoid cells, it does not appear to be associated with IL-4R on RCC cells. Even in the absence of common gamma chain IL-4 and IL-13 were able to up-regulate intracellular adhesion molecule-1 antigen on RCC cells. These data suggest that the interaction of IL-13 with IL-4R does not involve gamma c and IL-13R itself may be a novel subunit of the IL-4R.

Cross Reactions↗
PubMed1995

Circularly permuted interleukin 4 retains proliferative and binding activity.

In human interleukin 4(IL-4), the carboxyl and amino termini of the 129 amino acid hormone are close to each other and this region is believed to be important for binding to the IL-4 receptor (IL-4r). We constructed plasmids encoding circularly permuted IL-4 mutants with the peptide Gly-Gly-Asn-Gly-Gly (GGNGG) joining the carboxyl to the amino terminus and with new amino and carboxyl termini elsewhere. Mutant IL-4(38-37) is composed of IL-4 residues 38-129 GGNGG and 1-37. Mutant IL-4(105-104) is composed of IL-4 residues 105-129, GGNGG and 1-104. IL-4(38-37) and IL-4(105-104) were purified from E. coli to near homogeneity and retained 50-100% of the binding and proliferative activity of IL-4, and in addition retained the ability to upregulate CD23 on Burkitt's lymphoma cells. Circular dichroism studies indicated that the tertiary structures of both IL-4(38-37) and IL-4(105-104) were retained, with the former molecule most similar to native IL-4. We conclude that while both native termini of IL-4 may be near its binding site, neither is required to be free for optimum activity.

Amino Acid Sequence↗
PubMed1995

Systemic treatment with interleukin-4 induces regression of pulmonary metastases in a murine renal cell carcinoma model.

Advanced metastatic renal cell carcinoma has been shown to be responsive to immunotherapy but the response rate is still limited. We have investigated the therapeutic potential of systemic interleukin-4 (IL-4) administration for the treatment of pulmonary metastases in the murine Renca renal adenocarcinoma model. Renca cells were injected iv in Balb/c mice to induce multiple pulmonary tumor nodules. From Day 5, Renca-bearing mice were treated with two daily injections of recombinant murine IL-4 for 5 consecutive days. IL-4 treatment induced a significant reduction in the number of lung metastases in a dose-dependent manner and significantly augmented the survival of treated animals. Immunohistochemistry studies, performed on lung sections, showed macrophage and CD8+ T cell infiltration in the tumor nodules 1 day after the end of IL-4 treatment. The CD8 infiltration increased by Day 7 after IL-4 treatment. Granulocyte infiltration was not detectable. To clarify further the role of the immune system in IL-4 anti-tumor effect, mice were depleted of lymphocyte subpopulations by in vivo injections of specific antibodies prior to treatment with IL-4. Depletion of CD8+ T cells or AsGM1+ cells abrogated the effect of IL-4 on lung metastases, whereas depletion of CD4+ T cells had no impact. These data indicate that CD8+ T cells and AsGM1+ cells are involved in IL-4-induced regression of established renal cell carcinoma.

Animals↗
PubMed1995

Constitutive expression of human immunodeficiency virus type 1 tat gene inhibits interleukin 2 and interleukin 2 receptor expression in a human CD4+ T lymphoid (H9) cell line.

Human immunodeficiency virus (HIV-1) tat, a trans-activator of the HIV long terminal repeat, is essential for HIV replication and causes inhibition of antigen-mediated T cell proliferation. To understand the mechanism of inhibition of T cell proliferation, we have investigated the regulation of IL-2 production and its receptor expression on a human CD4+ T lymphoid cell line (H9) transfected with HIV-1 tat gene. When cells were activated by mitogens, as compared to control cells, a significant decrease in both IL-2 mRNA and protein was observed in tat-transfected cells. Similarly, mitogen-induced IL-2R alpha and IL-2R beta mRNA and surface expression of IL-2R alpha and IL-2R beta chains were also significantly decreased in tat-transfected cells compared to control cells. Only IL-2 receptor density was decreased; the affinity of the ligand for the receptor appeared to be unchanged. In contrast to our previous studies with B-lymphoblastoid cell line (Puri RK and Aggarwal BB: Cancer Res 1992; 52:3787-3790), IL-4R expression was unaltered by HIV tat transfection in the H9 T cell line, indicating a cell type-specific phenomenon. Owing to the central role of IL-2 immunoregulation, our data suggest that immunosuppressive effects of HIV-1 tat may be mediated at least in part through the inhibition of both IL-2 production and IL-2 receptor expression.

CD4-Positive T-Lymphocytes↗
PubMed1995

Intralesional treatment of established murine primary renal tumor with interleukin-4: localized effect on primary tumor with no impact on metastases.

In an effort to develop new strategies for immunotherapy of metastatic renal cell carcinoma, we investigated the therapeutic potential of interleukin-4 in a visceral renal tumor using the murine Renca renal adenocarcinoma model. Renca cells were implanted underneath the renal capsule of Balb/c mice to induce a primary tumor that spontaneously metastasized to several organs. Established primary renal tumors 4 to 6 mm. in diameter were treated by intralesional administration of recombinant murine interleukin-4 (IL-4). This treatment caused a marked inhibition of the primary tumor growth but had little effect on the progression of metastases in the liver, mesentery and lungs. Immunohistochemistry studies performed on renal tumor sections showed a macrophage infiltration that became predominant 7 days after IL-4 treatment. CD8+ T cells were also observed at the periphery and within the tumor. These data suggest that IL-4 mediated a potent antitumor effect when administered intralesionally although its effects remained localized with no impact on metastases at distant sites. Interleukin-4 antitumor activity seems to be mediated by recruitment of macrophages and T cells in the tumor.

Animals↗
PubMed1995

Suramin blocks binding of interleukin-4 to its receptors on human tumor cells and interleukin-4-induced mitogenic response.

Suramin, a polysulphonated naphthylurea, has antiproliferative, anticancer, and anti-HIV activities and has been shown to prevent binding of a variety of growth factors to their respective receptors. In the current study we have investigated the effects of suramin on binding of interleukin-4 (IL-4) to its receptors and IL-4-induced biological response. We found that suramin prevented the binding of 125I-labeled IL-4 to its receptor in a dose-dependent manner. The concentration of suramin that caused 50% inhibition of IL-4 binding (IC50) ranged between 55 and 70 microM. This effect was observed on two human renal cell carcinoma cell lines (PM-RCC and WS-RCC), a human T lymphoma cell line (H9), and a human premyeloid cell line (TF-1). Cross-linking experiments provided direct evidence that suramin prevented binding of 125I-labeled IL-4 to its receptors. Radiolabeled IL-4 specifically cross-linked with major proteins of approximately 145 and 65-70 kDa in both PM-RCC and H9 cell lines. Suramin prevented cross-linking to both affinity cross-linked IL-4 binding proteins. Gel filtration results indicated that suramin caused aggregation of 125I-labeled IL-4. Suramin had a cytostatic rather than cytotoxic effect on H9 cells, and it inhibited IL-4-induced proliferation of TF-1 cells. These data indicate that suramin may be a useful drug in the abrogation of IL-4-induced effects, and this property should be further explored in IL-4-mediated pathologic states.

Antigens, CD↗
PubMed1995

Urodynamic study of children with voiding problems.

Twenty high risk children aged 5-12 years with various voiding problems were studied prospectively by urodynamics to evaluate the function of their urinary bladder and its continence mechanism. None of them had neuropathic bladder or any obstruction distal to bladder neck. Fourteen out of twenty (70%) had abnormal findings on urodynamics evaluation; 8 (40%) had non-neurogenic neurogenic bladder (NNNB); 3 (15%) had small capacity hypertonic bladder (SCHB); 2 (10%) had atonic bladder (AB) and 1 (5%) had hyperreflexic bladder (HB). We conclude that urodynamic abnormalities are as frequent in high risk Indian children as they are in developed countries. The high risk children should be subjected to urodynamic studies more frequently than being done hitherto and be directed to proper therapeutic modality.

Analysis of Variance↗