| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
Departments of Pathology [K. D., C. S., D. M.] and Medicine [S. A. K.], Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
and is mediated by a specific amino acid sequence (104–123) in the ß-domain of the pVHL. In the present study, the amino acid sequence (104–123) of the pVHL was conjugated to the protein transduction domain of HIV-TAT protein (TATFLAGVHL-peptide) to facilitate entry into cells, and we demonstrate that this amino acid region of VHL is sufficient to block proliferation and invasion of 786-O renal cancer cells in vitro. Furthermore, daily i.p. injections with the TATFLAGVHL peptide retarded and, in some cases, caused partial regression of renal tumors that were implanted in the dorsal flank of nude mice. Treatment with this peptide also inhibits the invasiveness of renal tumors. A 56% decrease in the proliferative index in tumors treated with the TATFLAGVHL-peptide versus control-peptide-treated mice was observed. Taken together, these results show the novel importance of a 20-amino acid sequence of the ß-domain of the VHL gene product capable of inhibiting tumor growth and invasion. These results lay the foundation for a unique approach toward treating RCCs using this small-molecular-weight peptide fused to the TAT-sequence, which may, in the future, be used alone or in conjunction with other therapies. |
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Sporadic clear cell renal carcinoma often exhibits a functional inactivation of the pVHL (5, 6, 7, 8, 9, 10, 11)
. This tumor suppressor gene is a Mr-30,000 protein encoded by chromosome 3p25–26 and has been reported to be a potent down-regulator of several hypoxia-inducible genes including VPF/VEGF and hypoxia inducible factor-1
(12)
. pVHL has also been implicated in the regulation of the cell cycle regulatory protein p27 and the cellular ubiquitination machinery (13, 14, 15, 16, 17)
. The crystal structure of the pVHL published recently revealed a ß-sheet structure referred to as ß-domain in the NH2 terminus of pVHL and an -domain consisting of -helices in the COOH-terminus of pVHL (18)
. The amino acid region 152–171 within the -domain of VHL is found to be a highly mutated region in 6patients having VHL-associated RCC (10)
. This region is found to bind with elongin B and C and CUL-2, a member of the cullin family of proteins, and is thought to form a complex that closely resembles the yeast E3 type ubiquitin ligase complex (13
, 14
, 16
, 19, 20, 21)
. It was later shown that this complex can indeed exhibit E3 ligase activity (17)
. Thus, one of the major functions of pVHL is its role in the ubiquitination machinery that is involved in the degradation of a broad variety of cellular proteins.
RCC is dependent on IGF-I for its growth and development. Experimental results showed that inhibiting this IGF-I-mediated signaling abolishes or delays the progression of a variety of tumors in animal models (22, 23, 24)
. Previously, we had shown that the pVHL, apart from its role in the ubiquitination machinery, could also inhibit IGF-IR-mediated signaling in RCC (25)
. This inhibition is accomplished by the ability of the 104–123 amino acid region of the ß-domain of the VHL molecule to block the interaction between the cytoplasmic domain of the IGF-IR and its downstream signaling molecule PKC, thereby blocking PKC kinase activity. Interestingly, this region is also found to be mutated in patients having VHL-associated RCC (8)
. Intervention in the IGF-IR signaling pathway may ultimately affect cell proliferation, differentiation, and apoptosis, which might in turn affect tumor development (22, 23, 24
, 26)
. Thus, using a peptide coding for the 104–123 amino acid sequence of the pVHL may inhibit tumor development.
To evaluate the effects of this specific region of pVHL on tumor characteristics, we have used the TAT-mediated peptide delivery system to facilitate the entry of a peptide corresponding to the 104–123 region of the pVHL into renal cancer cells. The TAT protein is derived from HIV (27) and is capable of entering the cell in a receptorless fashion (28) . Although the mechanism of this protein transduction is unclear, it is believed that the TAT protein targets the lipid bilayer component of the cell membrane and is rapidly internalized in a concentration-dependent manner. An 11-amino acid region of TAT protein, the PTD, when fused to a broad spectrum of proteins regardless of size and function, potently facilitates entry into a majority of mammalian cell types (28, 29, 30) . Using this peptide delivery system, we tested our hypothesis that the 104–123 amino acid region of VHL, conjugated with the HIV-TAT sequence, can inhibit the growth of renal tumor.
|
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Cell Culture.
The human renal carcinoma cell line (786-O, ATCC CRL-1932; American Type Culture Collection, Manassas, VA) was maintained in DMEM containing 10% FBS (Fisher Scientific).
Cell Proliferation Assays.
RCC 786-O cells were plated in 24-well plates (1 x 103 cells/well) and starved for 16 h in DMEM (Fisher Scientific) containing 0.1% FBS. The TATFLAGVHL(104–123)-peptide, TATFLAGVHL(152–171)-peptide, or TATFLAG-peptide (Genemade Synthesis, Inc., San Francisco, CA), were added at different concentrations in DMEM containing 0.1% FBS, whereupon 0.5 µCi/ml [3H]thymidine (NEN, Boston, MA) was added to each well and incubated for an additional 4 h. Cells were then washed three times with cold PBS and subsequently precipitated with 5% ice-cold TCA for 15 min, rinsed twice with 75% ethanol, and solubilized in 0.1 M NaOH for scintillation counting (Perkin-Elmer Wallac, Inc., Gaithersburg, MD).
Invasion Assays.
Forty µl of an 8-mg/ml Matrigel solution (Fisher Scientific) was overlaid on the upper surface of the transwell chambers with a diameter of 6.5 mm and a pore size of 8 µm (Corning CoStar Corporation, Cambridge, MA). The Matrigel was allowed to gel by incubating the plates for 
4 h at room temperature. DMEM (0.6 ml) media containing 0.5 µM IGF-I used as a chemoattractant (Sigma) was then added to the bottom chamber of the transwells. 786-O renal carcinoma cells were trypsinized and then resuspended in DMEM media containing 0.2% BSA and preincubated for 1 h with medium containing 40 µM of TATFLAGVHL(104–123)-peptide, TATFLAGVHL(152–171)-peptide, or the TATFLAG-peptide. Thereafter, 2 x 104 cells in a volume of 100 µl of medium were added to the upper chamber of each well. Cells were then incubated for 5 h at 37°C in a CO2 incubator. Cells that remained in the upper chamber were removed by gently scraping with a cotton swab. Cells that had invaded through the filter were fixed in 100% methanol and then stained with 0.2% crystal violet dissolved in 2% ethanol. Invasion was quantitated by counting the number of cells on the filter using bright-field optics with a Nikon Diaphot microscope equipped with a 16-square reticle (1 mm2). Three separate fields were counted for each filter.
Slot Blot, Western Blot, and Immunocytochemical Analysis.
786-O renal carcinoma cells were cultured to near confluency in 100-mm2 tissue culture-treated plates in DMEM containing 10% FCS. The cells were then incubated overnight with either TATFLAGVHL-peptide or TATFLAG-peptide at a concentration of 20 µM. Cells were then washed twice with ice-cold PBS, lysed with ice-cold lysis buffer [50 mM Tris (pH 7.5), 1% NP40, 150 mM NaCl, 1 mM Na3VO4, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 0.5% aprotinin, and 2 mM pepstatin A) and incubated for 10 min on ice. Cell lysates were centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant from each sample was either transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) for slot blotting using Bio-Rad slot blot apparatus or was separated by SDS-PAGE. Size-separated proteins were transferred (Trans-Blot SD; Bio-Rad) to a polyvinylidene difluoride membrane (NEN). For immunodetection, membranes were blocked in washing buffer containing PBS [0.13 M NaCl and 10 mM sodium phosphate (pH 7.4)] and 0.1% Tween 20 with 4% milk or BSA and incubated in washing buffer with either mAb anti-phosphoMAP kinase (0.2 µg/ml; New England BioLabs), mAb anti-MAP kinase antibody (0.2 µg/ml; New England BioLabs), or polyclonal anti-FLAG antibody (4.4 µg/ml; Sigma). The secondary antibodies used were goat antirabbit and goat antimouse IgG coupled to horseradish peroxidase (Pierce, Rockford, IL). Immunolabeling was detected by chemiluminescence (Pierce). To detect the internalization of TAT peptides, the 786-O renal carcinoma cells were grown on coverslips in a 24-well plate. Cells were treated with different TAT peptides and then incubated for 12 h. The cells were washed twice in PBS and fixed in fresh 4% paraformaldehyde for 10 min at room temperature, rinsed in PBS, permeabilized when appropriate in PBS+0.1% Tween 20 for 15 min, rinsed, and then blocked in PBS, 0.1% BSA, and 0.1 M glycine. The cells were then incubated with anti-FLAG IgG for 1 h, rinsed, and incubated with the biotinylated goat antichicken IgG and rinsed. Staining was performed with the Vectastain ABC Elite kit with diaminobenzidine as the peroxidase substrate. Coverslips where then mounted in crystal mount (Biomedia, Foster City, CA).
RCC Tumor Model.
Male Harlan Sprague Dawley nude mice 6–8 weeks of age (National Cancer Institute, Bethesda, MD) were given s.c. injections in the right dorsal flank with 3 million 786-O RCC cells suspended in 100 µl PBS. Tumors appeared 2 weeks after injection. Tumor size was measured using calipers, and tumor volume was calculated using the formula, 
. After the tumors reached x200 mm3, the animals were randomized to receive daily i.p. injections with either TATFLAG-peptide or TATFLAGVHL(104–123)-peptide (2 nmoles/animal).
Surgical Specimens.
At the end of the treatment period, mice were killed by CO2 narcosis and tumors were excised. Tissues were embedded in OCT compound and frozen in liquid nitrogen pending subsequent analysis. All procedures were performed under hospital Institutional Review Board-approved protocols.
Immunohistochemical Staining.
All antibodies were diluted in PBS containing 0.1% BSA. Six-µm-thick serial cryosections were fixed in ice-cold acetone and rinsed in PBS. IgG binding sites were blocked with normal rabbit serum for 1 h at room temperature. Sections were incubated with the primary antibody for 60 min, rinsed, and incubated with 3% H202 in methanol for 15 min to deplete endogenous peroxidase. After rinsing, sections were incubated with biotinylated rabbit antirat IgG (7.5 µg/ml) for 30 min. Staining was performed with the avidin-biotin complex technique, using the Vectastain ABC Elite kit (Vector Laboratories) with 3,3'-diaminobenzidine as the peroxidase substrate. Finally, the sections were counterstained with Mayer’s hematoxylin. Sections were dehydrated in ethanol and cleared in xylene and then mounted with permount.
Quantification of Proliferation and Apoptosis.
Six-µm-thick cross sections through the entire tumor were subjected to PCNA or TUNEL staining according to the manufacturer’s instructions and lightly counterstained with Mayer’s hemotoxylin. The PCNA- or TUNEL-positive nuclei were manually counted in the entire section. Thereafter, four fields of vision were photographed, the total number of nuclei were counted manually, and the average number of nuclei/unit area was calculated. Photographs of the entire cross section were digitized using a Scan Jet 11CX scanner (Hewlett Packard, Grenoble, France). The area of the entire cross-section was calculated in mm2 using NIH imaging software. The total number of nuclei in the entire cross-section was calculated by extrapolating the average number of nuclei/mm2 multiplied by the area of the cross-section. The proliferation index and apoptosis index were calculated as the number of positive nuclei divided by the total number of nuclei. To access heterogeneity with regards to proliferation within an individual tumor, sections were taken from three different areas of the tumor and the proliferative index was determined as described above.
Quantification of Vessel Density.
To calculate vessel density, sections were stained with a monoclonal antibody against PECAM-1 but were not counterstained. Cross-sections were photographed at x2.5 and digitized using a Scan Jet 11CX scanner (Hewlett Packard). The digitized images were analyzed using NIH imaging software and processed as follows. Images were filtered using an edge enhancement filter. Subsequently, a threshold level was introduced. All pixels with values below this threshold were excluded from subsequent analysis. Thereafter, the number of remaining pixels, corresponding to the area of PECAM-1 stain, was calculated and converted to mm2. The area of PECAM-1-positive stain then was divided by the total area of the cross section, resulting in the percentage of tissue area occupied by PECAM-1-positive endothelium. Results from the TATFLAGVHL-peptide versus TATFLAG-peptide groups were analyzed statistically using Student’s t test and were considered significant at P < 0.05.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
. Slot-blot analysis of cell lysates derived from cells that had been incubated with the TATFLAGVHL or TATFLAG-peptide and then immunoblotted with an anti-FLAG antibody was performed to insure entry of these peptides into the cells (Fig. 2A)
. Fig. 2B
shows the immunostaining of the FLAG peptide of the TATFLAGVHL(104–123)-treated RCC only in permeabilized cells (Fig. 2B
, i), but not in nonpermeabilized cells (Fig. 2B
, ii) or cells that had not been treated with any peptide (data not shown). Therefore, the peptides detected in cell lysates as well as in immunostaining represents peptide that has been internalized.
|
|
. Incubating 786-O cells with a TATFLAGVHL construct containing the elongin-binding region of the VHL molecule (152–171 amino acid region) had no significant inhibitory effect on cell proliferation (Fig. 3A)
. Furthermore, inhibition of tyrosine phosphorylation of MAP kinase was observed in cells treated with the TATFLAGVHL(104–123)-peptide, but not the TATFLAG-peptide (Fig. 3B)
. Phosphorylated MAP kinase is an essential downstream molecule and nodal point for several signal transduction pathways that lead to cell proliferation. Thus, the results suggest that the 104–123 amino acid region of the VHL molecule is sufficient for blocking IGF-1-induced RCC proliferation in vitro.
|
. Incubating 786-O cells with the TATFLAGVHL(152–171)-peptide had no significant inhibitory effect on cell invasiveness (Fig. 4)
. Thus, our results suggest that the 104–123 amino acid sequence of the pVHL is sufficient to inhibit IGF-I-induced invasion of renal carcinoma cells in vitro.
|
200 mm3. At this point, either TATFLAGVHL(104–123)-peptide or TATFLAG-peptide (2 nmol/mouse) was injected i.p. once daily for 11 days, and tumor volume was measured daily. The mice were subsequently killed for histopathological analysis. In contrast to the TATFLAG-peptide-treated group, tumor growth was arrested after approximately 4 days, and a limited regression of tumor size was noted beginning 7 days after initiation of treatment in the TATFLAGVHL(104–123)-peptide-treated mice (Fig. 5)
. Tumor size after 11 days of peptide treatment was significantly smaller in TATFLAGVHL(104–123)-treated mice compared with TATFLAG-peptide-treated mice: 357 ± 156 mm2 and 839 ± 194 mm2, respectively (Table 1)
.
|
|
. Both peptides were localized to the tumor cells. Stromal cells also stained for the peptides, albeit with a lesser intensity. Therefore, these results suggest that the peptide effectively penetrated the entire tumor mass and was able to enter individual cells within the tumor. Tumors derived from untreated mice did not show immunoreactivity to the antibody recognizing the FLAG sequence (Fig. 6, A and C)
.
|
30%. Furthermore, heterogeneity in the proliferative index between different regions of the tumor biopsy was 28%. Thus, the proliferative index as determined through the total number of PCNA-positive nuclei divided by the total number of cell nuclei in a cross section of an individual tumor is representative of the entire tumor’s proliferative index. The apoptotic index did not differ significantly between tumors in TATFLAGVHL(104–123)-peptide-treated mice as compared with mice treated with the TATFLAG-peptide, amounting to 0.49% and 0.59%, respectively (Table 1)
. In contrast, a 56% decrease of the proliferative index was observed in tumors of TATFLAGVHL(104–123)-peptide treated mice as compared with that of TATFLAG-peptide treatment (Table 1)
. A 56% reduction in the proliferative index would theoretically result in 4-fold larger tumors in TATFLAG-peptide- versus TATFLAGVHL(104–123)-peptide-treated mice after 50 cell cycles, indicating that an actual treatment benefit can be achieved from a 56% reduction in the proliferative index (Fig. 7)
. Furthermore, no difference in vascular density between TATFLAGVHL(104–123)-peptide- and TATFLAG-peptide-treated tumors was observed (Table 1)
. Compared with TATFLAG-peptide-treated tumors, TATFLAGVHL(104–123)-treated tumors exhibited an increase in cellular density and a decrease in the size of the stromal compartment. This suggests that the TATFLAGVHL(104–123)-peptide may have an effect on the nonendothelial component of the stromal compartment (Table 1)
. The observed decrease in proliferative index with a constant apoptotic index might explain the observed regression of tumors in the TATFLAGVHL(104–123)-peptide- versus TATFLAG-peptide-treated mice, as has been described in other animal tumor models (31)
. In conclusion, the TATFLAGVHL(104–123)-peptide inhibits tumor growth by inhibition of tumor cell proliferation in vivo, in agreement with our in vitro observations.
|
. In contrast, tumors in 10 of 10 TATFLAG-peptide-treated mice were firmly adherent to the underlying muscle (Fig. 6F)
and/or skin, which was indurated and hyperemic (not shown). Tumors in the TATFLAG-peptide-treated mice exhibited marked invasion deep into the underlying muscle layer (Fig. 6H)
and, in two mice, invasion into the spinal column was observed (data not shown). Corresponding tissue planes allowing the radical removal of tumors in TATFLAGVHL(104–123)-peptide-treated mice could not be found in TATFLAG-peptide-treated mice. Histologically, islands of tumor cells were seen invading the skin and underlying muscle in TATFLAG-peptide-treated mice (data not shown). In contrast, consistent with the macroscopic findings, no invasion of tumor into surrounding normal tissue was observed in TATFLAGVHL(104–123)-peptide-treated mice (data not shown). Furthermore, in tumors in mice treated with the TATFLAG-peptide, but not the TATFLAGVHL(104–123)-peptide, invasion of tumor cells into blood vessels was frequently observed (Fig. 6I
; Ref. 32
). Autopsy revealed no gross distant metastatic disease in the lung, liver, kidney, and intestine in either the TATFLAGVHL(104–123)-peptide-treated mice or the TATFLAG-peptide-treated mice. These results show that the 104–123 amino acid region of the VHL molecule delivered as the TATFLAGVHL(104–123)-peptide construct inhibits RCC invasion in vivo. |
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
The biological activities of IGFs are, for the most part, mediated through IGF-IR. IGF-I activates an intrinsic tyrosine kinase activity in the receptor, resulting in its autophosphorylation and subsequently by the presentation of its substrate-binding sites (33)
. Substrates containing either the Src homology domain or phosphotyrosine binding domain can interact with IGF-IR to promote various downstream signal transduction cascades that ultimately lead to cell proliferation, differentiation, anti-apoptosis and, in pathological conditions, tumor development (26
, 34
, 35) . Importantly, embryonic fibroblasts established from IGF-IR(-/-) mice show resistance to transformation induced by different oncogenes, growth factor receptors, and viral proteins that can be reversed by reconstitution with wild-type IGF-IR (26)
. Also, blocking of IGF-IR signaling by any of several strategies (antisense, dominant negative, or neutralizing antibody against IGF-IR) abolishes or delays the progression of a variety of tumors in animal models (22, 23, 24)
. Recent studies have shown that PKC plays an important role in IGF-IR-mediated cell proliferation and transformation (36)
. It associates with IGF-IR and gets tyrosine-phosphorylated, resulting in increased activity. It has also been demonstrated that the ATP-binding mutant of PKC can inhibit the transforming ability of IGF-I (36)
. Because the amino acid region 104–123 in the ß-domain of the pVHL has the potential to block the IGF-IR-mediated downstream signaling pathway (25)
, we hypothesized that introducing this region into the renal cancer cell would restore the antiproliferative and anti-invasive properties of the pVHL. Therefore, we used the TAT-mediated peptide delivery system by which VHL(104–123)-peptide was delivered efficiently into the renal cancer cells both in the tissue culture as well as in the animal model (Figs. 2
and 6
, B and D). We found that in both in vitro and in vivo systems, this TATFLAGVHL(104–123)-peptide can inhibit renal cancer cell proliferation as well as invasion (Figs. 3A
, 4
, and 6
; Table 1
). Interestingly, using the same peptide delivery system, the 152–171 amino acid region of the pVHL, known for its ubiquitination function (13
, 14
, 16
, 19 , 20)
, is unable to inhibit either renal cancer cell proliferation or invasion (Figs. 3A
and 4
). This finding thus suggests a different function of VHL apart from its role in the ubiquitination pathway.
VHL-associated tumors are highly vascularized. Both VHL disease-associated and sporadic renal cell carcinomas overexpress VPF/VEGF, a potent angiogenic factor, and its receptors, suggesting that these genes may be VHL target (37, 38, 39)
. We and others have demonstrated that VPF/VEGF is indeed a target for the VHL tumor suppressor gene product (39, 40, 41)
. Interestingly, when tumor-bearing mice were treated with TATFLAGVHL-peptide(104–123), no difference in vascular density was observed as compared with that of the control peptide-treated mice, which might indicate that the TATFLAGVHL(104–123)-peptide is not primarily inhibiting tumor growth by targeting angiogenesis (Table 1)
. Future studies are in progress to define the region(s) of VHL that might be accountable in the suppression of the angiogenesis response of the tumors.
The ramifications of these TATFLAGVHL(104–123)-peptide-induced effects on renal cancer biology and treatment are several-fold. (a) Tumor invasion of blood vessels and surrounding tissues are important prognostic factors that are routinely evaluated in the histology of RCCs. Thus, inhibition of this process would theoretically improve the prognosis of these patients. (b) RCC is noted for its resistance to radiation and chemotherapies. It also shows only a modest palliative response in a subset of patients to immunomodulation therapies. For this reason, the only treatment alternative that offers the possibility of cure is radical surgical excision. The inhibition of invasion and reduction in size after TATFLAGVHL(104–123)-peptide treatment would therefore all be factors that would potentiate the chances of complete surgical excision of the tumor and/or its metastases. (c) Treatment of solid tumors with drugs is hampered by the relative inability of drugs to penetrate the tumor. This is believed to be, to a large extent, attributable to increased interstitial pressure, composition of the tumor stroma, and poor blood perfusion. Therefore, using a low-molecular-weight drug such as VHL peptide conjugated with the TAT sequence would be advantageous for the treatment of solid tumors because tumor penetration would be predicted to be less of a problem. In support of this notion, penetration of the VHL peptide was homogeneous throughout the tumors, and it entered a great majority of the tumor cells and, to a lesser extent, the stromal cells (Fig. 6)
.
In summary, our studies show the importance of the 20-amino acid sequence in the ß-domain of pVHL for its tumor growth- and invasion-inhibitive property. This region of VHL peptide is not related to the well-known function of VHL, where it acts as a part of ubiquitination machinery (13 , 14 , 16 , 19 , 20) . Here, we also describe a novel approach by using a part of a tumor suppressor gene product to inhibit the tumor growth and invasiveness of an existing tumor. Therefore, our findings will help to formulate a potential anticancer drug for the treatment of RCC in the near future.
|
FOOTNOTES |
|---|
1 Supported by NIH Grant CA78383 (to D. M.). C. S. was supported by a grant from the Swedish Cancer Foundation and Konung V:s 80-arsfond. D. M. is a Eugene P. Schonfeld National Kidney Cancer Association Medical Research Awardee. 
2 To whom requests for reprints should be addressed, at Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, RN270H, Boston, Massachusetts 02215. Phone: (617) 667-7853; Fax: (617) 667-3591; E-mail: dmukhopa{at}caregroup.harvard.edu
3 The abbreviations used are: RCC, renal cell carcinoma; VHL, von Hippel-Lindau; pVHL, VHL gene product; VPF, vascular permeability factor; VEGF, vascular endothelial growth factor; IGF-I, insulin-like growth factor-I; IGF-IR, IGR-I receptor; PKC, protein kinase c ; PTD, protein transduction domain; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; mAb, monoclonal antibody; MAP, mitogen-activated protein; PCNA, proliferating cell nuclear antigen.
Received 8/ 1/00. Accepted 1/15/01.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
is an important signaling molecule in insulin-like growth factor I receptor-mediated cell transformation. Mol. Cell. Biol., 18: 5888-5898, 1998.This article has been cited by other articles:
|
|
 |
|
  K. L. Wu, H. Miao, and S. Khan JAK kinases promote invasiveness in VHL-mediated renal cell carcinoma by a suppressor of cytokine signaling-regulated, HIF-independent mechanism Am J Physiol Renal Physiol, December 1, 2007; 293(6): F1836 - F1846. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Noguchi, V. Ropars, C. Roumestand, and F. Suizu Proto-oncogene TCL1: more than just a coactivator for Akt FASEB J, August 1, 2007; 21(10): 2273 - 2284. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zhou, J. Fan, and J.-T. Hsieh Inhibition of Mitogen-Elicited Signal Transduction and Growth in Prostate Cancer with a Small Peptide Derived from the Functional Domain of DOC-2/DAB2 Delivered by a Unique Vehicle. Cancer Res., September 15, 2006; 66(18): 8954 - 8958. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Nakamura, P. Abreu-e-Lima, Y. Awakura, T. Inoue, T. Kamoto, O. Ogawa, H. Kotani, T. Manabe, G.-J. Zhang, K. Kondo, et al. Clusterin Is a Secreted Marker for a Hypoxia-Inducible Factor-Independent Function of the von Hippel-Lindau Tumor Suppressor Protein Am. J. Pathol., February 1, 2006; 168(2): 574 - 584. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hiromura, F. Okada, T. Obata, D. Auguin, T. Shibata, C. Roumestand, and M. Noguchi Inhibition of Akt Kinase Activity by a Peptide Spanning the {beta}A Strand of the Proto-oncogene TCL1 J. Biol. Chem., December 17, 2004; 279(51): 53407 - 53418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. G. Kaelin Jr. The Von Hippel-Lindau Tumor Suppressor Gene and Kidney Cancer Clin. Cancer Res., September 15, 2004; 10(18): 6290S - 6295S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Wang, H. Zeng, P. Wang, S. Soker, and D. Mukhopadhyay Neuropilin-1-mediated Vascular Permeability Factor/Vascular Endothelial Growth Factor-dependent Endothelial Cell Migration J. Biol. Chem., December 5, 2003; 278(49): 48848 - 48860. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. G. Kaelin Jr. The von Hippel-Lindau Gene, Kidney Cancer, and Oxygen Sensing J. Am. Soc. Nephrol., November 1, 2003; 14(11): 2703 - 2711. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Fittipaldi, A. Ferrari, M. Zoppe, C. Arcangeli, V. Pellegrini, F. Beltram, and M. Giacca Cell Membrane Lipid Rafts Mediate Caveolar Endocytosis of HIV-1 Tat Fusion Proteins J. Biol. Chem., September 5, 2003; 278(36): 34141 - 34149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Murata, N. Tajima, Y. Nagashima, M. Yao, M. Baba, M. Goto, S. Kawamoto, I. Yamamoto, K. Okuda, and H. Kanno Von Hippel-Lindau Tumor Suppressor Protein Transforms Human Neuroblastoma Cells into Functional Neuron-like Cells Cancer Res., December 1, 2002; 62(23): 7004 - 7011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. MUKHOPADHYAY and H. ZENG Involvement of G Proteins in Vascular Permeability Factor/ Vascular Endothelial Growth Factor Signaling Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 275 - 284. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
, TATFLAG; 
, TATFLAGVHL(104–123); 
, TATFLAGVHL(152–171). B, proteins from cell lysates derived from 786-O cells that had been incubated with either TATFLAG or TATFLAGVHL-peptides were separated by SDS gel electrophoresis, transferred to polyvinylidene difluoride membranes, and subjected to immunoblotting with antibodies recognizing phosphoMAP kinase and MAP kinase. Phosphorylation levels of MAP kinase, but not total MAP kinase protein, is decreased in TATFLAGVHL-peptide-treated cells compared with control cells treated with TATFLAG-peptide and no peptide.
, TATFLAGVHL(104–123);
, TATFLAGVHL(152–171).
, TATFLAGVHL.
