A screen for genes that suppress loss of contact inhibition: identification of ING4 as a candidate tumor suppressor gene in human cancer

Abstract

We have devised a screen for genes that suppress the loss of contact inhibition elicited by overexpression of the protooncogene MYCN. The initial application of this screen detected nine distinctive suppressors within a representative human cDNA library. One of these genes was ING4, a potential tumor suppressor gene that maps to human chromosome 12p13. Ectopic expression of ING4 suppressed the loss of contact inhibition elicited by either MYCN or MYC but had no direct effect on cellular proliferation. Pursuing the possibility that ING4 might be a tumor suppressor gene, we found inactivating mutations in ING4 transcripts from various human cancer cell lines. In addition, we used comparative genomic hybridization to detect deletion of the ING4 locus in 10-20% of human breast cancer cell lines and primary breast tumors. Ectopic expression of ING4 attenuated the growth of T47D human breast cancer cells in soft agar. We conclude that ING4 is a strong candidate as a tumor suppressor gene.

Fig. 1.

A screen for genes that suppress the loss of contact inhibition induced by MYCN. (A) Schematic diagram of the screen. (B) Differential killing of cells with AZT/5-FU in an MYCN-dependent manner. Rat1A/MYCNER cells were grown to a confluent monolayer and incubated for 2 days in the presence or absence of 100 nM 4-OHT and/or 50 μM AZT/50 μM 5-FU. After 2 days, all drugs were withdrawn, and the cells were fed with fresh media every 3 days for 21 days. (C) ING4 suppresses the loss of contact inhibition elicited by MYCN. Rat1A/MYCNER/tTA cells containing pRevTRE-ING4 were grown in the presence or absence of 2 μM doxycycline for the duration of the assay. Confluent monolayers were subjected to 2 days of treatment in the presence or absence of 100 nM 4-OHT and/or 50 μM AZT/50 μM 5-FU.

Fig. 2.

ING4 in human cancer cell lines. (A) Expression of ING4. Northern blot analysis was performed on 5 μg of total cellular RNA by using ³²P-labeled full-length ING4 cDNA as probe. (B) Schematic diagram of the ING4 protein showing the locations of plant homeodomain (PHD) and nuclear localization signal (NLS). Arrows mark the mutations found in ING4 transcripts from human cancer cell lines.

Fig. 3.

Ectopic reconstitution of ING4 in T47D breast cancer cells. (A) Southern blot. Genomic DNA was digested with BamHI and analyzed by Southern blotting with ³²P-labeled full length cDNA of ING4 or the actin gene, ACTB,as hybridization probes. (B) CGH analysis of T47D (filled circles) and MDAmb231 (open squares) cell lines. Loss and gain of DNA are reflected in the relative copy number score on the y axis of the graph. Relative copy numbers were determined by the ratio of the test DNA to the reference DNA. Therefore, a -0.5 score indicates that the test DNA contained half the copy number compared with the reference DNA, reflecting the loss of one copy in the test DNA. As such, loss of two copies would score -1.0. Small arrows mark the location of BAC probes, RP11-272L6 at 5.1 Mb and RP11-59H1 at 12.6 Mb from telomere. A cytological banding of chromosome 12, the location of ING4, and the extent of deletion in T47D are shown. (C) Suppression of growth in soft agar. T47D cells infected with the retroviral constructs, ING4-IRES-GFP and IRES-GFP, were plated in soft agar and then incubated for 21 days. Colonies were counted with the aid of either light or fluorescent microscopy.

Fig. 4.

Genomic deletion of the ING4 locus in primary breast tumors. CGH analysis of three tumors, A (filled circles), B (sectored squares), and C (open squares), are shown in a graph. The extent of each deletion is represented with bars.