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Tumor Biology |
Renal Division, Department of Medicine [S. A., B. K., W. G., M. D., G. W., V. P. S.] and Department of Pathology [I. E. S.], Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3–4-fold) in 786-O RCC cells by decreasing the TGF-ß1 mRNA half-life. Exogenously added TGF-ß1 did not suppress the growth of 786-O cells in vitro, nor did the addition of neutralizing antibody (Ab) against TGF-ß have any effect. Indeed, 786-O cells were found to express no TGF-ß type II receptor protein, thus allowing them to escape from the negative growth control of TGF-ß1. In contrast to the in vitro data, neutralizing Ab to TGF-ß inhibited tumorigenesis and, in some cases, regressed established 786-O tumors in athymic mice. Immunohistochemistry for von Willebrand’s factor revealed a 3–4-fold lower tumor microvessel count in the mice treated with TGF-ß Ab compared to controls, suggesting that the Ab was inhibiting angiogenesis. Our findings indicate that TGF-ß1 is a novel target for the VHL tumor suppressor and that antagonizing its paracrine action may provide novel avenues for treatment of RCCs as well as other tumors that secrete TGF-ß1. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RCC is the most common cancer of the kidney, occurring in over 27,000 individuals in the United States each year, and it is responsible for over 11,000 deaths annually (18)
. The treatment of RCC remains frustrating to the oncologist, and locally unresectable, metastatic disease has a dismal prognosis. There is a tremendous need to understand the basic biology of RCC and develop better therapeutic options. Most sporadic and hereditary RCCs (VHL -disease associated) show mutation and/or loss of both copies of the VHL gene (18)
. pVHL is lost in early atypical cysts, suggesting that pVHL might play a gatekeeper role in RCC development, analogous to the APC gene product in colon cancer (19
, 20)
. VHL -disease-associated tumors are typically hypervascular and target genes identified to date include VEGF, TGF-
, and platelet-derived growth factor ß, all of which have proangiogenic effects (21, 22, 23, 24)
. TGF-ß1 is another gene that is significantly involved in angiogenesis (25)
. Although TGF-ß1 has been found to be elevated in RCCs (5, 6, 7
, 12
, 14)
, there has been no link to date with the VHL tumor suppressor, and no functional role has been ascribed to TGF-ß1 for RCC growth in vivo.
We describe here that TGF-ß1 is a novel target gene for pVHL and that pVHL regulates the TGF-ß1 gene at the posttranscriptional level. Furthermore, we provide evidence that antagonizing the effects of TGF-ß1 suppresses RCC in vivo through an antiangiogenic mechanism.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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VHL), were gifts from W. Kaelin and have been described previously (26)
. 786-O FLAG VHL, 786-O Neo, 786-O HA VHL, and 786-O HA VHL were grown DMEM with 10% FBS and supplemented with G418. The wt 786-O and CCL-64 (mink lung epithelial cells) cells were grown in DMEM with 10% FBS.
RNA Extraction and Northern Blot Analysis
These experiments were done as described previously (23)
. The probes used were a TGF-ß1 2.1-kb EcoRI fragment (phTGTB-2 from the American Type Culture Collection) and a 600-bp PCR-amplified fragment from human actin cDNA.
ELISA Analysis of TGF-ß1 Secretion
ELISA of cell culture supernatants obtained from confluent cultures was performed using the manufacturer’s recommended procedures (Quantakine kit; R&D Systems). All samples and standards were run in triplicate. Color intensity was measured at 450 nm using a spectrophoretic plate reader. Activation of latent TGF-ß1 to immunoreactive TGF-ß1 was performed according to the manufacturer’s recommendations, and all samples were measured before (active TGF-ß1) and after activation (latent TGF-ß1).
Western Blot Analysis
Cells were grown to 70–80% confluence and lysed in lysis buffer as described previously (22
, 23)
. Equal amounts of cell lysates (50 µg), as determined by the Bradford assay, were loaded. Abs included TGF-ß type II receptor-specific Ab (Santa Cruz Biotechnology, Santa Cruz, CA), actin Ab (Santa Cruz Biotechnology), and VHL Ab (a gift from W. Kaelin).
Nuclear Run-on in Vitro Transcription Assay
Nuclei isolation and in vitro transcription were performed as described previously (22
, 23)
. Probes were either gel-purified inserts from plasmids or generated by PCR. The TGF-ß1 probe used (2 kb) contained the entire coding region of TGF-ß1, including 779 bp of the 5' untranslated sequence and 216 bp of the 3' untranslated sequence. The amount of sample hybridized to the TGF-ß1 probe was normalized by dividing the TGF-ß1 signal by that of GAPDH within each experiment.
Actinomycin D Experiments
These experiments were performed as described previously (22)
. Briefly, subconfluent cells were exposed to 5 µg/ml actinomycin D (Sigma) for 0–24 h. Total RNA was isolated from 786-O cells, and the signal on Northern blot analysis was determined for TGF-ß1 after normalization for RNA loading and transfer using the 28S band on the transferred membrane.
Assessment of Growth Properties after Exposure to TGF-ß1
DNA synthesis was studied in triplicate by [3H]thymidine incorporation into DNA. Cells (2 x104) were plated in 24-well plates in DMEM with 0.5% FBS. At 24 h after plating, the medium was replaced with or without TGF-ß1 (1 or 10 ng/ml; R&D Systems) and anti-TGF-ß1,-ß2,-ß3 monoclonal Ab (10 µg/ml; Genzyme) for 48 h. In vitro and in vivo neutralization of this TGF-ß Ab has been published previously (27, 28, 29)
. During the last 4 h, 2 µCi of [3H]-thymidine (DuPont New England Nuclear) were added. Cells were then washed twice in PBS and lysed in 5 N NaOH. [3H]-Thymidine incorporation of the cells was assessed in a liquid scintillation beta counter (Beckman).
RCC Tumor Model
The 6–8-week-old NIH-3 nude beige xid mice (Harlan Sprague Dawley) and nude mice (Harlan Sprague Dawley) were given s.c. injections in the right flank of 3 million 786-O cells in a 100-µl volume. Tumors appeared approximately 2 weeks after implantation. Tumor size was measured using calipers, and tumor volume was calculated using the following formula: volume = width2 x length/2. Tumor volume ranged from 150–200 mm3. The animals were randomized into two groups of four mice each with comparable tumor size. Mice were then injected i.p. with 100 µg of monoclonal anti-TGF-ß1,-ß2,-ß3 Ab (the same Ab used in vitro and described above) or control nonspecific IgG (Sigma) on alternate days. When treatment was terminated, animals were sacrificed, and tumors from each mouse were removed and divided into two pieces: one piece was fixed in 10% formalin and paraffin-embedded; and the other was cryopreserved in OCT. The paraffin-embedded specimens were stained with H&E and Masson’s trichrome stain and evaluated by light microscopy.
Immunohistochemistry
Factor VIII Staining/Microvessel Count.
Six-µm-thick sections from cryopreserved tumor specimens were stained for endothelial cells using Factor VIII Ab (DAKO), using a standard immunoperoxidase technique as described previously (30)
. The microvessel count was done according to the method of Delahunt et al. (31)
. After the area of highest neovascularization was identified under low power (x10), individual microvessels were counted on four adjacent high-power fields (x40), and the mean microvessel count was determined. Any area showing positive staining for Factor VIII was considered to be a countable vessel, regardless of whether or not a distinct lumen was visible. In the tumors in which the microvasculature formed a dense network, each distinct branch was interpreted as a single vessel.
Proliferation Index/Apoptotic Index.
The Apotag plus in situ apoptosis detection kit (Oncor, Gaithersburg, MD) was used to measure the apoptotic index in paraffin sections of tumors, following the manufacturer’s instructions. The proliferative index was determined by using a monoclonal Ab against Ki-67, clone MIB-1 (Immunotech, Westbrook, ME), on paraffin sections of tumors, followed by the standard immunoperoxidase technique. For both of the indices, the areas with the highest cellular staining were selected, and four fields (each field, 0.65 mm2) were studied. For each field, the number of positive and negative staining tumor cells was quantified. These were combined to create a percentage of positive cells per unit area (2.6 mm2).
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RESULTS |
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, IL-6, epidermal growth factor receptor, and TGF-ß1) were analyzed by Northern blot in the 786-O RCC cell lines. The 786-O RCC cells, derived from a patient with sporadic metastatic RCC, lack wt pVHL (26)
. Strikingly, TGF-ß1 mRNA levels were repressed 4-fold in 786-O RCC cell lines stably transfected with wt VHL (786-O HAVHL) compared with levels in the same cell lines transfected with an empty vector (786-O Neo) or transfected with a mutant VHL lacking aa 116–213 (786-O HA VHL; Fig. 1A)
. To address the possibility of clonal variation, we confirmed the findings in wt 786-O cells and in 786-O FLAG VHL cells generated in our laboratory (Fig. 1A)
. Levels of protein expression from the stably transfected constructs were assessed by Western blot (Fig. 1B)
. ELISA confirmed that VHL-transfected cells secreted 3–4-fold less latent and active TGF-ß1 protein in the culture supernatant (Fig. 1C)
. These data indicate that both TGF-ß1 mRNA and protein are repressed by pVHL, indicating that TGF-ß1 mRNA is a target for pVHL.
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. Similarly, mutant VHL did not alter the transcription of the TGF-ß1 gene (Fig. 2A)
. Experiments performed in the presence of -amanitin showed an inhibition of TGF-ß1 transcription, indicating the specificity of our experimental conditions (Fig. 2A)
. A shorter TGF-ß1 probe (700 bp in the 5' untranslated region) gave similar results (data not shown). Our nuclear run-on data suggest that the regulation of TGF-ß1 by VHL is predominantly posttranscriptional, e.g., at the level of RNA splicing, nuclear export, or mRNA stability.
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VHL cells by measuring the decay of TGF-ß1 mRNA in the presence of the transcriptional inhibitor actinomycin D. The TGF-ß1 mRNA half-life was 11.48 h in VHL-transfected cells compared to 39.36 h in mutant VHL-transfected cells and 25.92 h in empty vector-transfected cells (Fig. 2B)
. Proof that actinomycin D was active during the early time points of the experiments came from probing the blots with a c-myc probe: the signal fell dramatically and was undetectable at 2 h (data not shown). These results suggest that pVHL destabilizes TGF-ß1 mRNA.
786-O RCC Cells Are Unresponsive to Exogenous TGF-ß1 in Vitro and Lack Functional TGF-ß Type II Receptor.
TGF-ß1 binds with high affinity to the type II receptor. This binding is followed by recruitment of the type I receptor and subsequent intracellular signal transduction (2)
. We checked whether the RCC cell lines were growth responsive to exogenous TGF-ß1. We found that the growth of 786-O cells was unaffected by exogenous TGF-ß1 at 1 and 10 ng/ml (Fig. 3A)
. In addition, neutralizing Ab against TGF-ß (which was used later in vivo) did not have any effect. As controls, mink lung epithelial cells (CCL-64) that have functional TGF-ß receptors were found to be growth responsive to TGF-ß1, and neutralizing Ab against TGF-ß reversed the TGF-ß1-induced growth suppression (Fig. 3A)
. We then examined the expression of the TGF-ß type II receptor protein in 786-O cells. Western blot analysis revealed no TGF-ß type II receptor protein in 786-O Neo and 786-O HAVHL cells. Mink lung epithelial cells expressing functional TGF-ß type I and II receptors were used as a positive control (Fig. 3B)
. These data suggest that the loss of type II receptor expression in 786-O cells is responsible for their unresponsiveness to TGF-ß1 in vitro.
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) or a control Ab were injected i.p. on alternate days for five doses. Animals treated with TGF-ß Ab showed a striking difference in tumor size when compared to controls (Fig. 4A)
after 10 days. Interestingly, one of the four tumors in the treated group regressed to a small nodule of <50 mm3 (data not shown). At the end of the experiment, the mice were sacrificed, and the tumors were examined histopathologically. There was no difference between treated and control groups on H&E stain (no infiltrating cells noted) and on Masson’s trichrome stain. Areas of patchy necrosis in the rapidly growing control tumors were noted (data not shown). Importantly, Factor VIII staining showed a 3–4-fold decrease in the number of microvessels in the treated group (Fig. 4, B and C)
. Furthermore, the proliferative index of the treated group was 3-fold lower than in the controls, with no change in the apoptotic index (Fig. 4C)
. Experiments repeated in athymic nude (nu/nu) mice (T-cell deficient mice with normal NK activity; n = 4 controls and 4 treated mice) gave very similar results, including one tumor regression (data not shown). These data suggest that the primary mechanism of tumor suppression through the use of TGF-ß neutralizing Ab is the inhibition of angiogenesis.
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. Both tumors regressed in the treated group over this time period but regrew rapidly upon cessation of therapy. Therapy was reinstituted when the mean tumor size was 280 mm3, and tumor growth was again suppressed with a second course of TGF-ß Ab. Collectively, these data suggest that antagonizing TGF-ß in vivo significantly inhibits RCC tumors in athymic mice, with regressions seen in 4 of 10 mice treated. Moreover, these tumors do not develop resistance to this therapy, consistent with the microvessel count data suggesting inhibition of angiogenesis as the therapeutic mechanism involved. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The human VHL gene encodes a 213-aa protein and is expressed in all tissues. VEGF, platelet-derived growth factor, and glucose transporter-1 were the first target genes identified for pVHL (21
, 23
, 24)
. We have recently found that TGF- is another target gene for pVHL (22)
. Here we report that both TGF-ß1 mRNA and protein levels are suppressed by wt VHL in two 786-O cell transfectants. These data provide the first connection between a tumor suppressor product and TGF-ß1. Given the biological actions of TGF-ß1, its up-regulation in cells with mutant VHL could promote tumor progression in multiple ways: by promoting angiogenesis; by enhancing stromal proliferation; by increasing collagenase and gelatinase b activity; and by suppressing the immune response to the tumor (detailed below). Indeed, the concomitant dysregulation in RCCs of TGF-, VEGF, and TGF-ß1, which are all VHL targets, may be particularly efficacious in this regard, because all have proangiogenic actions, and TGF-, in addition, can act to directly stimulate tumor growth through an autocrine loop. The mechanism of action of pVHL is poorly understood. It has been shown that pVHL interacts in vitro with the regulatory subunits elongin B and elongin C of the transcription elongation complex (elongin SIII); however, none of the target genes identified thus far, including TGF-ß1, appears to be regulated at the level of transcriptional elongation. Moreover, VHL interacts with Hs-Cul-2; its yeast homologue Cdc 53 is part of a ubiquitin protein ligase complex that targets cell cycle proteins for degradation by the ubiquitin proteolytic pathway (33
, 34)
. Collectively, these data point to a role for complexes containing pVHL, elongin B, elongin C, and Cul-2 to target certain proteins, perhaps RNA-binding proteins, involved in the degradation of target mRNAs (33)
. Interestingly, VEGF, TGF-, and TGF-ß1 are all regulated largely at the level of the mRNA half-life, and it will be interesting to investigate whether the pVHL-elongin-Cul-2 complex is involved in controlling their degradation through some common mechanism, especially because the mutant COOH terminus VHL truncation that lacks the elongin C binding site (VHL) did not repress either of these targets (this study and Refs. 21, 22, 23, 24
).
Lack of a functional type II receptor and associated rearrangements of the gene or aberrations in transcription or in posttranscriptional steps have been described in several human tumors, including gastric, colon, breast, and T-cell malignancies (8 , 35, 36, 37, 38, 39) , but not in RCC. In this report, we have shown that 786-O cells do not express the type II receptor protein and are functionally resistant to the TGF-ß signal transduction. Studies are in progress to characterize the nature of the genetic defect leading to the absence of the type II receptor protein in RCC.
TGF-ß1 is up-regulated in several cancers. To date, studies correlating prognosis with TGF-ß1 levels in different cancers have not been consistent, although inverse associations have been found in hepatic, renal, cervical, and prostate cancer (13, 14, 15, 16, 17 , 40) . Some cancer cells also secrete activated TGF-ß1 (41) ; however, RCCs predominantly secrete latent TGF-ß1 (7 , 12 , 17) . Moreover, when compared to primary cancers, cell lines derived from metastatic cancers can show greater TGF-ß1 production and resistance to its growth-inhibitory effect but increased collagenase activity (42) .
Multiple biological actions of TGF-ß1 in the etiology of cancer can be invoked. The proangiogenic effects of TGF-ß1 in potentiating tumor progression are likely to be important. Targeted disruption of either the TGF-ß1 gene or its type II receptor results in defective placental vasculogenesis (43
, 44)
. TGF-ß1 regulates other angiogenic molecules, such as VEGF, TGF-, and VEGF receptor flk-1 (1
, 45)
, and has very recently been shown to inhibit the generation of the antiangiogenic protein angiostatin via modulation of the plasminogen/plasmin system (46)
. Although TGF-ß1 inhibits endothelial cell proliferation in vitro, its overall effect in vivo is proangiogenic (25)
. An explanation of these seemingly discrepant results is that TGF-ß1 plays an important role in the resolution phase of angiogenesis by directly inhibiting endothelial cell growth and migration and reducing extracellular proteolysis. Stable transfection of TGF-ß1 confers a growth advantage on Chinese hamster ovary cells in vivo but not in vitro, accompanied by an increase in capillary density; local administration of neutralizing Ab to TGF-ß1 reduced both capillary density and tumor growth (47)
. However, a large quantity of Ab (5 mg) was used to neutralize the overexpressed TGF-ß1 produced by cancer cells, in contrast to the 100 µg of Ab used to antagonize endogenous TGF-ß1 in our experiments. TGF-ß1-mediated angiogenesis is contextual, i.e., in the presence of positive regulators such as VEGF and bFGF, an additive or synergistic angiogenic response is noted. Also, the effect of TGF-ß1 on endothelial cell function is concentration dependent, i.e., VEGF- and bFGF-induced capillary invasion in an in vitro three-dimensional model was dependent on the concentration of TGF-ß1 and was highest at 200–500 pg TGF-ß1/ml (25)
. These facts may explain why the neutralizing Ab against TGF-ß did not inhibit angiogenesis in a breast cancer model (48)
, whereas in the 786-O RCC model, VHL mutation leads to the up-regulation of multiple factors (VEGF, TGF-, and TGF-ß1) perhaps potentiating each other’s angiogenic effects.
Despite rapid progress in characterizing cell surface receptors for TGF-ß, little is known about the function of TGF-ß receptors in endothelial cells, and virtually nothing is known about the role of TGF-ß ligand-receptor interactions in the regulation of angiogenesis in vivo. Endothelial cells respond equally to TGF-ß1 and TGF-ß3 and respond poorly, if at all, to TGF-ß2, possibly due to the low affinity of endoglin (type III receptor) for TGF-ß2 in endothelial cells. Although the Ab we used is panspecific (against TGF-ß1, TGF-ß2, and TGF-ß3), the effect we have seen is probably mediated by the neutralization of TGF-ß1 because 786-O RCC cells do not express TGF-ß3 and express very low levels of TGF-ß2 mRNA (data not shown).
TGF-ß1 may also enhance carcinogenesis by suppressing the immune response (1 , 2 , 48) . In our nude beige mice RCC model, there are no T cells, B cells, and NK cells; hence, we believe that tumor suppression is predominantly a result of the antiangiogenic effect, as suggested by the microvessel count data. Experiments in syngeneic RCC models might elucidate an additional therapeutic benefit of helper and cytolytic T-cell activity and NK cell activity upon neutralization of TGF-ß1 action. In this context, antagonizing TGF-ß1 action may be particularly appropriate for the treatment of RCCs in man, because these tumors secrete numerous immunomodulatory cytokines in addition to TGF-ß, including IL-10 and IL-6 (14) . These cytokines diminish T-cell responses to the tumor, and neutralizing part of their activity may therefore show unusual benefit in renal cancer therapy.
Several additional features of our tumor data are worth highlighting. First, anti-TGF-ß therapy regressed established tumors. In 4 of 10 treated animals, tumors diminished to a size of 50 mm3, reminiscent of the rests seen after therapy with endostatin, an antiangiogenic COOH terminus 20-kDa fragment of collagen XVIII (49
, 50)
. As with endostatin, the tumors regrew quickly upon cessation of therapy and could again be treated successfully. These data demonstrate the lack of drug resistance but highlight the need for either chronic therapy or multimodality therapy (see below) to effect a cure. Second, on a molar basis, anti-TGF Ab was 
60x more effective than endostatin, based on published data in non-RCC (51)
as well as our studies with endostatin in RCC using the same 786-O xenograft model (52)
. This number could be further increased with a higher-affinity Ab, or one with improved pharmacokinetics. Third, because endostatin appears to inhibit the initiation phase of angiogenesis (cell proliferation and migration; Ref. 52
), whereas TGF-ß is important in the resolution phase, it is conceivable that endostatin in combination with methods to neutralize TGF-ß activity may be additive or synergistic. Similarly, TGF-ß1 neutralization in combination with chemotherapy, immunotherapy, or radiation makes eminent sense. Also, it is noteworthy that the acquisition of TGF-ß unresponsiveness correlates well with tumor progression (12
, 42
, 53)
, so that anti-TGF-ß therapy will be the most useful in advanced disease, when other modalities are less likely to be effective. Fourth, there is the possibility that TGF-ß1 induced by the process of surgery itself (54)
or by radiation (55)
may stimulate metastatic growth, especially if the metastatic lesions are resistant to the growth-inhibitory activity of TGF-ß (42)
. Along these lines, O’Reilly (56)
has shown that a combination of TGF-ß1 and bFGF administered systemically can cause the regrowth of small tumor rests after endostatin therapy. These data point to the potential importance of monitoring TGF-ß1 levels in a cancer patient and assessing tumor profiling of TGF-ß and its receptors and suggests the possible use of anti-TGF-ß therapy either at the time of primary tumor removal or at surgery in a patient with cancer in remission or before and peri radiation therapy. Counterweighing this would be the potential deleterious effects of such therapy on wound repair. Finally, other approaches to negating the effect of TGF-ß1 may be more efficacious or practical than the use of neutralizing Ab. Decorin, a proteoglycan known to bind TGF-ß (57)
, latency associated peptide, soluble TGF-ß receptors, antisense oligonucleotides, and TGF-ß peptide antagonists (58)
may serve this purpose.
In conclusion, we have shown that TGF-ß1 is a target for the pVHL and that repression of the TGF-ß1 message occurs predominantly at a posttranscriptional level. Identification of potential RNA-binding proteins and destabilizing elements in the TGF-ß1 mRNA will help elucidate the function of pVHL. Whereas VHL mutations occur early in RCC development, an important secondary genetic event in the TGF-ß1 signaling pathway leads to the abrogation of TGF-ß type II receptor expression and resistance to the antiproliferative effects of TGF-ß1. Moreover, we have shown that the biological significance of elevated TGF-ß1 in RCCs is to stimulate angiogenesis and that neutralizing TGF-ß activity can regress established RCCs without the development of drug resistance. Blocking the paracrine effects of TGF-ß1 may provide novel treatment strategies for RCCs and other cancers that secrete TGF-ß1.
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FOOTNOTES |
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1 Supported in part by a NIH RO1 grant (to V. P. S.), a NIH training grant (to S. A.), and a grant from the National Kidney Foundation (to B. K.). 
2 To whom requests for reprints should be addressed, at Renal Division, Dana 517, Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. Phone: (617) 667-2105; Fax: (617) 667-7843; E-mail: vsukhatm{at}bidmc.harvard.edu
3 The abbreviations used are: TGF, transforming growth factor; RCC, clear cell renal carcinoma; VHL, von Hippel-Lindau; Ab, antibody; pVHL, VHL gene product; FBS, fetal bovine serum; wt, wild-type; aa, amino acid(s); VEGF, vascular endothelial growth factor; NK, natural killer; IL, interleukin; bFGF, basic fibroblast growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, hemagglutinin.
Received 11/13/98. Accepted 3/ 3/99.
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R. S. Muraoka-Cook, N. Dumont, and C. L. Arteaga Dual Role of Transforming Growth Factor ss in Mammary Tumorigenesis and Metastatic Progression Clin. Cancer Res., January 15, 2005; 11(2): 937s - 943s. [Abstract] [Full Text] [PDF]
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 M. I. Zhou, H. Wang, R. L. Foy, J. J. Ross, and H. T. Cohen Tumor Suppressor von Hippel-Lindau (VHL) Stabilization of Jade-1 Protein Occurs through Plant Homeodomains and Is VHL Mutation Dependent Cancer Res., February 15, 2004; 64(4): 1278 - 1286. [Abstract] [Full Text] [PDF]
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T. Massfelder, H. Lang, E. Schordan, V. Lindner, S. Rothhut, S. Welsch, P. Simon-Assmann, M. Barthelmebs, D. Jacqmin, and J.-J. Helwig Parathyroid Hormone-Related Protein Is an Essential Growth Factor for Human Clear Cell Renal Carcinoma and a Target for the von Hippel-Lindau Tumor Suppressor Gene Cancer Res., January 1, 2004; 64(1): 180 - 188. [Abstract] [Full Text] [PDF]
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 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]
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W.-M. Miao, W. Lin Seng, M. Duquette, P. Lawler, C. Laus, and J. Lawler Thrombospondin-1 Type 1 Repeat Recombinant Proteins Inhibit Tumor Growth through Transforming Growth Factor-{beta}-dependent and -independent Mechanisms Cancer Res., November 1, 2001; 61(21): 7830 - 7839. [Abstract] [Full Text] [PDF]
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N. Takahashi, A. Haba, F. Matsuno, and B. K. Seon Antiangiogenic Therapy of Established Tumors in Human Skin/Severe Combined Immunodeficiency Mouse Chimeras by Anti-Endoglin (CD105) Monoclonal Antibodies, and Synergy between Anti-Endoglin Antibody and Cyclophosphamide Cancer Res., November 1, 2001; 61(21): 7846 - 7854. [Abstract] [Full Text] [PDF]
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 J. Lawler, W.-M. Miao, M. Duquette, N. Bouck, R. T. Bronson, and R. O. Hynes Thrombospondin-1 Gene Expression Affects Survival and Tumor Spectrum of p53-Deficient Mice Am. J. Pathol., November 1, 2001; 159(5): 1949 - 1956. [Abstract] [Full Text] [PDF]
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 S. A. KARUMANCHI, L. JIANG, B. KNEBELMANN, A. K. STUART-TILLEY, S. L. ALPER, and V. P. SUKHATME VHL tumor suppressor regulates Cl-/HCO3- exchange and Na+/H+ exchange activities in renal carcinoma cells Physiol Genomics, April 2, 2001; 5(3): 119 - 128. [Abstract] [Full Text] [PDF]
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 J. I. Izawa and C. P.N. Dinney The role of angiogenesis in prostate and other urologic cancers: a review Can. Med. Assoc. J., March 1, 2001; 164(5): 662 - 670. [Abstract] [Full Text] [PDF]
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Y.-H. Shiao, J. H. Resau, K. Nagashima, L. M. Anderson, and G. Ramakrishna The von Hippel-Lindau Tumor Suppressor Targets to Mitochondria Cancer Res., June 1, 2000; 60(11): 2816 - 2819. [Abstract] [Full Text] [PDF]
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 A. L. Harris von Hippel-Lindau Syndrome: Target for Anti-Vascular Endothelial Growth Factor (VEGF) Receptor Therapy Oncologist, April 1, 2000; 5(90001): 32 - 36. [Abstract] [Full Text]
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M. Los, S. Zeamari, J. A. Foekens, M. F. B. G. Gebbink, and E. E. Voest Regulation of the Urokinase-type Plasminogen Activator System by the von Hippel-Lindau Tumor Suppressor Gene Cancer Res., September 1, 1999; 59(17): 4440 - 4445. [Abstract] [Full Text] [PDF]
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 A. V. Ivanova, S. V. Ivanov, A. Danilkovitch-Miagkova, and M. I. Lerman Regulation of STRA13 by the von Hippel-Lindau Tumor Suppressor Protein, Hypoxia, and the UBC9/Ubiquitin Proteasome Degradation Pathway J. Biol. Chem., April 27, 2001; 276(18): 15306 - 15315. [Abstract] [Full Text] [PDF]
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P. Devarajan, M. De Leon, F. Talasazan, A. R. Schoenfeld, E. J. Davidowitz, and R. D. Burk The von Hippel-Lindau Gene Product Inhibits Renal Cell Apoptosis via Bcl-2-dependent Pathways J. Biol. Chem., October 26, 2001; 276(44): 40599 - 40605. [Abstract] [Full Text] [PDF]
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