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Mechanism of suppression of malignancy in hybrids between Y79 retinoblastoma and NIH3T3 cells
1Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami School of Medicine, FL 33136.
Abstract:
Hybrids were prepared between Y79 retinoblastoma (RB) and nonmalignant NIH3T3 cells and studied to confirm the presumed recessiveness of RB. Twenty hybrids containing both of the dominant gene markers, pSV2neo and pSV2GPT, initially transfected into the parent cells were isolated. All of the hybrids showed a fibroblastic morphology and anchorage-dependent growth. None of the tested hybrids or the parent NIH3T3 cells showed growth in soft agar; the parent RB cells showed a 15% growth in soft agar. The results indicate suppression of the malignant phenotype in the hybrids, confirming the recessiveness of the malignant phenotype of RB at the cellular functional level. Karyotyping of selected hybrids and the parent cells indicated a cumulative representation of all of the Y79 chromosomes in the hybrids, excluding loss of a specific Y79 chromosome causing the suppression of malignancy. Northern analysis of RNA from the hybrids demonstrated the mRNA of the reported putative mouse RB gene, consistent with the complementation of the Y79 RB gene defect by the normal mouse RB gene in the hybrids. Such a complementation may be a factor in the suppression of the malignant phenotype. Interestingly, the abnormal Y79 RB mRNA was absent in the hybrids, suggesting a possible negative feedback control by the normal mouse RB gene product.
Insights
Retinoblastoma (RB) cell malignancy is recessive. Hybrid cells formed by combining Y79 RB and NIH3T3 cells suppressed the malignant phenotype, confirming RB
Area of Science:
- Cell Biology
- Genetics
- Cancer Research
Background:
- Retinoblastoma (RB) is a malignant tumor of the retina.
- The genetic basis and cellular mechanisms of RB malignancy are not fully understood.
- Investigating RB cell hybrids can elucidate the genetic control of malignancy.
Purpose of the Study:
- To confirm the presumed recessiveness of retinoblastoma (RB) at the cellular level.
- To investigate the genetic and molecular mechanisms underlying RB malignancy suppression in hybrid cells.
Main Methods:
- Creation of hybrid cells by fusing Y79 retinoblastoma (RB) cells with nonmalignant NIH3T3 cells.
- Selection of hybrids using dominant gene markers (pSV2neo and pSV2GPT).
- Phenotypic analysis (morphology, anchorage dependence, soft agar growth), karyotyping, and Northern blot analysis of RNA.
Main Results:
- Hybrid cells exhibited nonmalignant fibroblastic morphology and anchorage-dependent growth.
- Suppression of malignant phenotype, including loss of soft agar growth, was observed in hybrids.
- Northern analysis revealed complementation of the Y79 RB gene defect by the normal mouse RB gene and absence of abnormal Y79 RB mRNA.
Conclusions:
- The malignant phenotype of retinoblastoma (RB) is recessive at the cellular functional level.
- Complementation of the RB gene defect by a normal counterpart gene contributes to malignancy suppression.
- A potential negative feedback mechanism by the normal RB gene product may regulate RB gene expression.