Analysis of baseline and cisplatin-inducible gene expression in Fanconi anemia cells using oligonucleotide-based microarrays

Abstract

BACKGROUND: Patients with Fanconi anemia (FA) suffer from multiple defects, most notably of the hematological compartment (bone marrow failure), and susceptibility to cancer. Cells from FA patients show increased spontaneous chromosomal damage, which is aggravated by exposure to low concentrations of DNA cross-linking agents such as mitomycin C or cisplatin. Five of the identified FA proteins form a nuclear core complex. However, the molecular function of these proteins remains obscure. METHODS: Oligonucleotide microarrays were used to compare the expression of approximately 12,000 genes from FA cells with matched controls. Expression profiles were studied in lymphoblastoid cell lines derived from three different FA patients, one from the FA-A and two from the FA-C complementation groups. The isogenic control cell lines were obtained by either transfecting the cells with vectors expressing the complementing cDNAs or by using a spontaneous revertant cell line derived from the same patient. In addition, we analyzed expression profiles from two cell line couples at several time points after a 1-hour pulse treatment with a discriminating dose of cisplatin. RESULTS: Analysis of the expression profiles showed differences in expression of a number of genes, many of which have unknown function or are difficult to relate to the FA defect. However, from a selected number of proteins involved in cell cycle regulation, DNA repair and chromatin structure, Western blot analysis showed that p21waf1/Cip1 was significantly upregulated after low dose cisplatin treatment in FA cells specifically (as well as being expressed at elevated levels in untreated FA cells). CONCLUSIONS: The observed increase in expression of p21waf1/Cip1 after treatment of FA cells with crosslinkers suggests that the sustained elevated levels of p21waf1/Cip1 in untreated FA cells detected by Western blot analysis likely reflect increased spontaneous damage in these cells.

Figure 1

p16 and p300 protein expression in FA and control cell lysates.10 μg protein from indicated cell extracts were immunoblotted with p16, β-tubulin, and p300 specific antibodies, as indicated. Extracts were from HSC536 stably transfected with empty vector (p; lanes 1) or with vector expressing FANCC (C; lanes 2), and from HSC72 stably transfected with vector expressing FANCA (A; lanes 3) or with empty vector (p; lanes 4). β-tubulin was used as additional loading control.

Figure 2

Cisplatin induced growth inhibition. VU450 cell lines were one hour pulse treated with indicated concentrations of cisplatin. Dotted line indicates the dosage used for gene expression experiments. VU450R; reverted (crosslinker resistant) cell line and VU450; FA (crosslinker sensitive) cell line.

Figure 3

Protein expression in FA and control cells treated with cisplatin. 10 μg protein from indicated cell extracts were immunoblotted with specific antibodies directed against indicated proteins; A) BAF170, CDC25a, PMS2, RGS2 and β-tubulin; B) p21^waf1/Cip1. Extracts were from HSC72 and HSC536 cells transfected with empty vector or with vector expressing correcting cDNA as indicated. Cells were one hour pulse treated with discriminating concentrations of cisplatin; HSC72 with 5 μM and HSC536 with 1 μM. β-tubulin was used as additional loading control.