Date of Award

1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pathology and Laboratory Medicine

College

College of Graduate Studies

First Advisor

Debra Hazen-Martin

Second Advisor

Gian Re

Abstract

Clear cell sarcoma of the kidney (CCSK) and Congenital mesoblastic nephroma (CMN) are both pediatric renal tumors that differ from Wilms' tumor based on their distinctive histopathologic characteristics and clinical behavior. CCSK is a very malignant tumor characterized by a propensity for bone metastases, contrasting the relatively benign course characteristic of the majority of Wilms' tumors. CMN, in contrast, is a tumor of the newborn usually diagnosed within the first three months of life that is generally cured with surgery alone. To investigate further the cellular and molecular relationships of CCSK, CMN, and Wilms' tumor, a nude mouse xenograft and cell culture system was established for both CCSK and CMN; the immunohistochemical, gene expression, and functional analysis of the p53 tumor suppressor in cell cycle control signal transduction was performed; and Northern blot analysis of genes relevant to normal nephrogenesis was examined. The histologic, ultrastructural, and molecular features of CCSK and CMN nude mouse xenografts, some of which have been passaged over several years, retained the characteristics of the primary tumors. Similarly, cell cultures derived from either primary tumors or mouse xenografts maintained cellular and molecular profiles consistent with findings for primary tumors. p53 mutation is a marker for poor prognosis in Wilms' tumors. p53 immunoreactivity was absent from a large panel of CCSK and CMN primary tumor specimens examined. However, immunohistochemical analysis of CCSK and CMN tumor cell lines surprisingly revealed p53 immunoreactivity in both CMN cell lines but in none of the CCSK cells examined suggesting that p53 was mutated in the benign CMN tumors. Functional analysis of p53 in the same CCSK and CMN cell culture system using a DNA damaging agent cis­ diamminedicholroplatinum (100 µM) demonstrated the presence of functional p53 as determined by the upregulation of p2l Waf-1/Cip-1 mRNA and G1 cell cycle arrest over an eight hour period. The histogeneses of CCSK and CMN is not known. CCSK and CMN primary tumor tissue was examined for mRNA expression of the genes WT-1, PAX-2, Pax-8, and tenascin- C, all genes relevant to normal kidney development. CCSK and CMN were generally negative for WT-1, PAX-2, and Pax-8 expression. CMN, but not CCSK tumor specimens, were positive for tenascin-C mRNA. To evaluate further the histogenesis of these tumors, cultured CCSK and CMN cells were treated with the differentiation agent all-trans retinoic acid (1µM) over three days. Induction of several genes were noted during this period such as PAX-2 and tenascin. The expression of IGF-2, IGF-BP2, and p21waf-1/cip1 were also modulated by this treatment. There were no light microscopically evident changes in cellular morphology in either CCSK or CMN cells. However, transmission and scanning electron microscopic analysis of the cells demonstrated closer cellular juxtaposition and flattening. These studies indicate that the in vivo and in vitro model system for CCSK and CMN will be useful in further investigating the molecular pathological relationships and tumorigenic mechanisms in pediatric renal tumors. The apparent absence of p53 functional impairment in CCSK distinguishes this aggressive tumor type from anaplastic Wilms' tumors which often bear mutation of p53 and suggests an alternative genetic explanation for the poor prognosis of CCSK. Further, a deeper understanding of the gene expression profile of pediatric renal tumors and renal development will be useful in the diagnosis and treatment of CCSK, CMN, and Wilms' tumor. Both the CCSK and CMN xenografts and cell lines also have potential utility in understanding the pathobiology of the developing fetal kidney as well as other pathologies affecting both children and adults.

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