Affiliations of authors: M. Los, O. A. J. Kerckhaert, E. E. Voest (Laboratory of Medical Oncology, Division of Medical Oncology, Department of Internal Medicine), R. Zewald, H. K. Ploos van Amstel (Department of Medical Genetics), University Medical Center Utrecht, The Netherlands.
Correspondence to: Emile E. Voest, M.D., Laboratory of Medical Oncology, Division of Medical Oncology, Department of Internal Medicine, University Medical Center Utrecht, P.O. Box 85500, 3508 GA Utrecht, The Netherlands (e-mail: e.e.voest{at}digd.azu.nl).
von HippelLindau (VHL) disease is an autosomal dominant, inherited cancer syndrome that is characterized by extensively vascularized tumors, such as hemangioblastomas of the retina and central nervous system, renal cell carcinomas (RCCs), and pheochromocytomas (1). The VHL tumor suppressor gene was cloned in 1993 and is located at chromosome 3p253p26 (2). According to the two-hit hypothesis for tumor suppressor genes, inactivation of both alleles of the VHL gene leads to tumor formation. One copy of the VHL gene is mutated by inheritance, and the second hit causes inactivation of the remaining wild-type VHL gene (3).
Although the VHL protein is widely expressed in different organs and cell types, its expression could not be demonstrated in endothelial cells of normal tissue (4). Strong expression of the VHL protein, however, was observed in proliferating endothelial cells from placenta, lung metastases of pancreatic adenocarcinoma, the base of an esophageal ulcer, and sprouting microvessels of atherosclerotic lesions [(5); Los M: unpublished observations]. In human umbilical vein endothelial cells and in dermal microvascular endothelial cells, expression of VHL messenger RNA was detected by northern blot analysis (Los M: unpublished observations).
VHL disease-related tumors are highly vascularized and are composed predominantly of endothelial and tumor cells. To explain the extensive vascularization, numerous studies [(1,6) and references therein] have shown increased production of growth factors in VHL disease tumors. In RCCs and hemangioblastomas from VHL disease patients, a dramatic increase in the expression of vascular endothelial growth factor (VEGF) has been observed (7). VEGF is a potent mitogen for endothelial cells, and its expression is regulated by the VHL gene. However, in a recent study (8), we could not demonstrate an association between VEGF levels in ocular fluid and the presence of retinal angiomas. We, therefore, hypothesized that endothelial cells may also be prone to additional mutations that result in uncontrolled proliferation.
By use of tissue microdissection and techniques of molecular biology, loss of heterozygosity (LOH) has been found in the clear-cell renal lesions of VHL disease-related RCCs and in the stromal cell component of VHL disease-related cerebellar and retinal hemangioblastomas (911). The isolation of endothelial cells that are depleted of tumor and stromal cells is virtually impossible; thus, LOH in the vascular component of the tumor could not be studied with this method.
In this study, we used a different method to examine if LOH for the VHL gene occurs in endothelial cells of VHL disease tumors. We collected tissue samples of RCCs from surgical pathology specimens from four VHL patients (patients 14) who underwent nephron-sparing surgery. We also obtained normal kidney tissue adjacent to the tumor from patients 1 and 2. In addition, a mutational analysis on DNA samples from leukocytes of affected family members was performed.
All experiments were performed according to the guidelines of the Medical Ethics Committee of the University Medical Center Utrecht.
From the RCCs, we obtained single-cell suspensions of tumor-derived endothelial cells and the residual tumor cells as described earlier (12). Briefly, surgical tissue was minced and incubated for 1 hour at 37 °C with 1 mg/mL collagenase and 1 mg/mL dispase (Sigma Chemical Co., St. Louis, MO) in RPMI-1640 medium (Life Technologies, Inc. [GIBCO BRL], Rockville, MD) containing 20% human pooled serum. The tissue was sieved to remove large cell clumps and cell debris, and the single-cell suspension was washed and resuspended in the same medium. Cells were either stained directly after isolation with the endothelial cell-specific marker Ulex-biotin (ICN Pharmaceuticals, Inc., Costa Mesa, CA) and streptavidin-R-phycoerythrin conjugate (DAKO, Glostrup, Denmark) or stained after overnight culture in RPMI-1640 medium in a gelatin-coated culture flask for endothelial cells with diiodoacetylated low-density lipoprotein (DiI-Ac-LDL; Biomedical Technologies, Stoughton, MA). Subsequently, DiI-Ac-LDL-positive or Ulex-positive cells were sorted in a cell sorter (Becton Dickinson Immunocytometry Systems, San Jose, CA).
DNA was isolated from the cell suspensions after cell sorting by resuspending the cells in a DNA lysis buffer (i.e., 0.1 M TrisHCl [pH 8.5], 5 mM EDTA, 0.2 M NaCl, 0.2% sodium dodecyl sulfate, and 100 µg/mL proteinase K). The DNA was amplified by the polymerase chain reaction (PCR) by use of primers for the three exons of the VHL gene (13). The PCR-amplified fragment was subsequently sequenced directly by use of the forward primers for the three exons.
The germline mutations could be detected in the DNA samples of the normal kidney tissue (Table 1; patients 1 and 2). The germline mutation in patient 2 is a deletion of exons 1 and 2; as a result, we detected only the normal sequence of one allele in normal kidney tissue. The mutations of patients 1 and 2 were identical to the sequence of the DNA isolated from leukocytes of affected family members. In all four patients, the only mutations that we detected in the three exons of the entire VHL gene analyzed in DNA obtained from tumor-derived endothelial cells were the germline mutations detected in DNA obtained from the adjacent normal kidney (patients 1 and 2) or in the DNA of an affected family member (patients 14). The sequence of DNA derived from the tumor cells of patients 1, 3, and 4 showed predominantly the following mutations: base position 722T
A (corresponding to Val170Asp) in patients 1 and 3 and base position 713G
A (corresponding to Arg167Glu) in patient 4. This observation indicates that the second hit in tumor cells of patient 1, 3, and 4 is the loss of the wild-type allele. In patient 2, the germline mutation is a deletion of exons 1 and 2. Direct sequence analysis of the tumor cells of patient 2 showed that the second hit was a missense mutation at position 452 (452G
A, corresponding to Ser80Asn).
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The extensive vascularization of tumors seen in VHL disease, the normal phenotype of endothelial cells derived from these tumors, and the life-long risk of these patients for developing tumors make them excellent candidates for antiangiogenic therapy.
NOTES
Supported by grant NWO 920-03-024 from The Netherlands Organization for Scientific Research, University Medical Center Utrecht.
We thank Drs. A. W. Griffioen and A.-R. van der Vuurst de Vries for technical assistance.
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9
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Manuscript received December 15, 1999; revised July 21, 2000; accepted August 4, 2000.
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