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The inhibitor of growth (ING) genes (ING1-4) probably descend from tumour suppressor genes. ING] was the first to be identified and later isolated using an approach to detect genes whose expression is suppressed in cancer. The others were isolated through homology and similarity searches in human and mouse databases. All members contain a plant homeodomain involved in macromolecule recognition. Apart from the extensively studied ING], little is known about the number of transcripts encoded by the other members or their gene structure. ING1 encodes several differentially spliced mRNAs, which may produce a family of proteins. The most widely expressed protein isoform is [p33.sub.INGb1] which is involved in restriction of cell growth and proliferation, apoptosis, tumour anchorage independent growth, cellular senescence, maintenance of genomic stability, and modulation of cell cycle checkpoints. ING1 gene mutation is uncommon in cancer, although the subcellular localisation of [p33.sub.INGb1] may have an effect o n its function. The [p33.sub.INGb1] cellular compartmental shift from the nucleus to the cytoplasm may cause loss of normal cellular function, and may play a central role in the pathogenesis of several cancers.
Two major classes of tumour associated genes have been implicated in tumorigenesis: oncogenes and tumour suppressor genes. Inactivation, by loss or mutation, of tumour suppressor genes plays an essential role in the genesis of many tumours. (1) Tumour suppressor proteins negatively regulate cell growth through a variety of mechanisms controlling the cell cycle. (2) As long as these genetic elements are fully active, these genes serve as potent buffers against tumour progression, preventing the deregulation of normal growth control.
INHIBITOR OF GROWTH GENE FAMILY
The inhibitor of growth (ING) gene family, although newly recognised, is thought to be a part of this evolutionarily old family of putative tumour suppressor genes. The INGI gene was the first member of this family to be identified and was later described by Garkavtsev and colleagues early in 1996. (3) The family presently comprises the ING1, ING2 (ING1-L), [ING3, and ING4 (ING2) genes. (3-7) These genes are conserved between species including humans, mouse, yeast, and frog. (7-12) In addition, these genes seem to maintain a high degree of homology with each other, which ranges from 32% to 76%.
CLONING AND ISOLATION STRATEGY
Limited numbers of tumour suppressors have been identified, (1) mainly because of the recessive nature of tumour suppressors and the lahourious identification methods involved. The ING1 gene was isolated using a new approach designed particularly for the detection of genes whose expression is suppressed in cancer cells; that is, tumour suppressor genes. (3) The technique is based on subtractive hybridisation and the subsequent selection and isolation of transforming genetic suppressor element (GSE) fragments. These fragments are short cDNA sequences that are capable of promoting neoplastic transformation. GSE fragments are isolated from random cDNA expression libraries. A normal mammary epithelial cell line (184A1) and seven breast cancer cell lines (MCF-7, BT-474, Hs-578T, ZR-75, MD-MB468, MD-MB-435, and BT-20) were used to prepare these cDNA libraries. These GSE fragments act effectively as oncogenes in gene transfection techniques through blocking the activation of tumour suppressors. Therefore, transfecti on and expression of the antisense sequences of the tumour suppressor gene would block protein production of this gene, hence, promoting cellular growth. In contrast, transfection and expression of the sense sequence of the tumour suppressor gene would block cellular growth.
The other members of the ING gene family were isolated through homology and similarity searches in cDNA library databases of human and mouse origin.
LOCATION, STRUCTURE, AND TRANSCRIPTS ENCODED BY MEMBERS OF THE ING GENE FAMILY
ING1 has been mapped to chromosome 13 and locus 13q33-34. (13 14) ING2 (INGIL) has been mapped to chromosome 4 at position 4q35.l. (4) ING3 has been mapped to chromosome 7 and locus 7q31. (15) Finally, ING4 (ING2) has been mapped to chromosome X at position Xq13.1. (16)
The ING gene family belongs to a larger family of genes encoding proteins that contain structural motifs that are involved in the regulation of transcription. All members of this family contain a plant homeodomain (PHD) finger made of metal chelating residues arranged in the following order: four cysteine, one histidine, and three cysteine residues (C4HC3 ). (17) This PHD finger domain spans 50-80 amino acids (aa).
"Although the ING1 gene has been extensively studied, little is known about the number of transcripts encoded by the other ING gene family members or their gene structure"
ING1 has been identified in humans and has been found to encode a series of differentially spliced mRNAs, with variable initiation sites and usage of three distinct exons (1a, 1b. and 2), as shown in fig 1. (17-19) These mRNAs comprise ING1a, ING1b, and ING1c, (5 20 21) which if translated would produce a family of proteins [p47.sup.ING1a] (422 aa and 46 451 Da), [p33.sup.ING1b] (279 aa and 31 843 Da), [p27.sup.ING1b] (235 aa and 27 000 Da), and [p24.sup.ING1c] (210 aa and 23 656 Da). (5 18-21) However, the most widely expressed of these protein isoforms appears to be [p33.sup.ING1b]. (5 6 22) All these proteins share a common exon (exon 2), which contains the PHD finger motif. The ING1 gene has been also identified in the mouse, yeast, and frog. Because of a cloning error, early investigations into the expression and the function of the ING1 gene products mistakenly used [p24.sup.ING1c] instead of [p33.sup.ING1b]. (l7 20 23)
We have identified three potential nuclear localisation signals (NLS) in the common C-terminal region (exon 2) consisting of highly positively charged amino acids (such as lysine, proline, and arginine)--PNSKRS, PKEKK, and KKKKR--at amino acid positions 145-151, 176-181, and 185-190, respectively. (22) Others have also found several NLS at amino acid positions 146-165 and l76-210. (17 24) Furthermore, two nucleolar targeting sequences (NTS) were identified at …