Newly established human retinoblastoma cell lines exhibit an “immortalized” but not an invasive phenotype in vitro

Abstract

Retinoblastoma (RB), an intraocular childhood tumor occurring in a hereditary (mostly bilateral) or non-hereditary (unilateral) form, is associated with the inactivation of both alleles of a putative tumor suppressor gene (RB-I) located on chromosome 13q14. Both the process of RB development and the biological characteristics of RB cells are as yet poorly understood. We have established 7 new RBL lines (RBL13, RBL14, RBL18 and RBL30, derived from unilateral RB; and RBL7, RBL15 and RBL20, derived from bilateral RB). Southern blot analyses of restriction fragment length polymorphisms in DNA samples from 6 cell lines revealed loss of constitutional heterozygosity at one or several polymorphic loci on chromosome 13 in 4 cases. Gross deletions involving the RB-I locus and amplification of the N-myc gene were not detected in any of the RBL lines. The phenotypic properties of the RBL lines were analyzed in comparison with cells from the original RB tumors, with 4 RB lines established by others (RB383, RB355, RB247C3 and Y79) and with the adenovirus-EIA-transformed human retinoblast line HER-Xhol-CC2. It was found that RB tumors consist of phenotypically heterogeneous cell subpopulations with varying nutrient requirements and differentiation potential in vitro. All cell lines showed the typical characteristics of established ("immortalized") cells. In some cases, cells from original RB tumors or cell lines were able to form colonies when cell aggregates of 2-10 cells were suspended in semi-solid agar medium; however, anchorage-independent colonies never developed from single cells. Cell lines RBL13, RBL18, RB247C3, RB355, RB383 and Y79 were tested for invasion into embryonic chick heart fragments in vitro and found to be non-invasive. None of the RBL or RB lines were tumorigenic in nu/nu (T-) mice. Y79 cells (propagated in culture for many years) exhibited properties distinctly different from those of the other cell lines, and thus cannot be considered phenotypically representative of RB cells.

Changes in Retinoblastoma Cell Adhesion Associated with Optic Nerve Invasion

“…The primary tumor was maintained in culture at a high cell density, and within a few weeks, two distinct cell populations developed, adherent cells and nonadherent cells. This heterogeneity in cell adherence has been reported in virtually every description of primary human retinoblastoma cultures (10,21,26). The immortal-suspension cells from this patient were eventually called Weri1 cells, and the adherent cells were not maintained.…”

“…Interestingly, many of the studies on the isolation and characterization of retinoblastoma cell lines have described changes in cell adhesion over the course of culturing the samples (10,21,26). For example, it has been reported that Y79 retinoblastoma cells initially grow in clumps, but then they start to lose cell adhesion and grow as single cells (26).…”

“…A more recent effort to identify and characterize retinoblastoma cell lines improved the efficiency of establishing cell lines by using human fibroblast feeder layers during the primary culture period (10) and provided a more careful analysis of the cells during the early phases of growth in culture. These data support the idea that the Y79 cells have undergone significant changes and/or selection in culture that distinguish them from the primary human retinoblastoma.…”

“…These data support the idea that the Y79 cells have undergone significant changes and/or selection in culture that distinguish them from the primary human retinoblastoma. Unfortunately, the cell lines established by Griegel et al are not available from the ATCC, and due to their slow growth, these cells are much more difficult to work with in the laboratory (10).…”

Genomic gains on chromosome 1q in retinoblastoma: Consequences on gene expression and association with clinical manifestation

“…23 Cell lines were cultivated in DMEM with 15% FCS, 100 U penicillin/ml, 100 lg streptomycin/ ml, 4 mM L-glutamine, 50 lM ethandiol and 10 lg insulin/ml at 37°C, 10% CO 2 and 80% humidity. DNA was extracted from cells harvested from these cell lines using the protocols described above.…”