The farnesyl transferase inhibitor SCH 66336 induces a G(2) --> M or G(1) pause in sensitive human tumor cell lines.

SCH 66336 is a potent farnesyl transferase inhibitor (FTI) in clinical development. It efficiently prevents the membrane association of H-ras, but not K- or N-ras. Yet, in soft agar, it reverts the anchorage-independent growth of human tumor cell lines (hTCLs) harboring H-ras, K-ras, and N-ras mutations, implying that blocking farnesylation of proteins besides ras may be responsible for this effect. Experiments show that SCH 66336 altered the cell cycle distribution of sensitive human tumor cells in two distinct ways. Most sensitive hTCLs accumulated in the G(2)-->M phase after the FTI treatment, but those with an activated H-ras accumulated in G(1) phase, suggesting that the biological effects induced by FTIs in cells with an activated H-ras are distinct from other sensitive cells. A careful genotypic comparison of the hTCLs revealed that those cells with wild-type p53 are especially sensitive to the FTIs. In these cells p53 and its downstream target gene p21(Cip1) are induced after treatment with SCH 66336 for 24 h. These data suggest that cell cycle effects, either G(1) or G(2)-->M accumulation, and p53 status are important for mediating the effects of FTIs on tumor cells.

Farnesyl transferase inhibitors block the farnesylation of CENP-E and CENP-F and alter the association of CENP-E with the microtubules.

Human tumor cell lines that are sensitive to the effects of farnesyl transferase inhibitors accumulate in G(2) --> M (except for cells with an activated Ha-ras that accumulate in G(1)). A search for CAAX box proteins from Swiss-Prot revealed more than 300 peptides. Of these, the centromeric proteins CENP-E and CENP-F are preferentially expressed during mitosis and are implicated as mediators of the G(2) --> M checkpoint. Experiments performed here show that peptides from the COOH-terminal CAAX box of CENP-E and CENP-F are substrates for farnesyl transferase but not geranylgeranyl transferase-I. Although both proteins are prenylated in the human tumor cell line DLD-1, their prenylation is completely inhibited by the farnesyl transferase inhibitor, SCH 66336. Immunohistochemical data with the lung carcinoma cell line, A549, showed that preventing the farnesylation of CENP-E and CENP-F by treatment with the farnesyl transferase inhibitor SCH 66336 does not affect their localization to the kinetochores. However, the presence of farnesyl transferase inhibitors alters the association between CENP-E and the microtubules. Our results imply that the inhibition of CENP-E farnesylation results in the alteration of the microtubule-centromere interaction during mitosis and results in the accumulation of cells prior to metaphase.

Misexpression of disrupted HMGI architectural factors activates alternative pathways of tumorigenesis.

Cancer arises from aberrations in the genetic mechanisms that control growth and differentiation. HMGI-C and HMGI(Y) are members of the HMGI family of architectural factors expressed in embryonic or undifferentiated cells and highly associated with transformation. Translocations of 12q13-15 in lipomas (fat cell tumors) disrupt HMGI-C and fuse its DNA-binding domains to novel transcriptional regulatory domains. This study shows that in a rare, karyotypically distinct group of human lipomas, rearrangements of 6p21-23 produce internal deletions within HMGI(Y). Activation of the rearranged alleles leads to expression of aberrant HMGI(Y) transcripts in differentiated adipocytes. A molecular analysis of these transcripts demonstrates that fusion of HMGI DNA-binding domains to putative transcriptional regulatory domains was not necessary for lipoma formation. However, such fusions may facilitate tumor development because activation of the wild-type HMGI allele, normally required for tumorigenesis, is bypassed in lipomas which express chimeric HMGI proteins. We hypothesize that HMGI misexpression in a differentiated cell is a pivotal event in benign tumorigenesis, and the molecular pathway of tumor development depends upon the precise nature of HMGI disruption.

Translocation breakpoints upstream of the HMGIC gene in uterine leiomyomata suggest dysregulation of this gene by a mechanism different from that in lipomas.

Uterine leiomyomata are the most common pelvic tumors in women and are the indication for more than 200,000 hysterectomies annually in the United States. Rearrangement of chromosome 12 in bands q14-q15 is characteristic of uterine leiomyomata and other benign mesenchymal tumors, and we identified a yeast artificial chromosome (YAC) spanning chromosome 12 translocation breakpoints in a uterine leiomyoma, a pulmonary chondroid hamartoma, and a lipoma. Recently, we demonstrated that HMGIC, which is an architectural factor mapping within the YAC, is disrupted in lipomas, resulting in novel fusion transcripts. Here, we report on the localization of translocation breakpoints in seven uterine leiomyomata from 10 to > 100 kb upstream of HMGIC by use of fluorescence in situ hybridization. Our findings suggest a different pathobiologic mechanism in uterine leiomyomata from that in lipomas. HMGIC is the first gene identified in chromosomal rearrangements in uterine leiomyomata and has important implications for an understanding of benign mesenchymal proliferation and differentiation.

Genomic characterization of human HMGIC, a member of the accessory transcription factor family found at translocation breakpoints in lipomas.

HMGIC, a member of the HMGI family of high mobility group proteins, is disrupted in 3/3 lipomas characterized by 12q14-q15 rearrangements. To define the genomic structure of the HMGIC gene, YACs from the HMGIC locus were identified and subcloned in lambda FIXII, and genomic lambda clones containing the HMGIC exons were isolated. The HMGIC gene consists of five exons that span at least 60 kb and encodes a 4.1-kb transcript. The coding region is 330 bp, the 5'UTR is 854 bp, and the 3'UTR is 2966 bp. Intron 3, which separates the DNA-binding domains from the acidic domain, is unusually large ( > 25 kb) and is the site of disruption in lipomas with 12q14-q15 translocations. The genomic organization of HMGIC should further our understanding of the 12q14-q15 region, where other neoplasms of mesenchymal origin are also mapped.

Mutation responsible for the mouse pygmy phenotype in the developmentally regulated factor HMGI-C.

Growth is one of the fundamental aspects in the development of an organism. Classical genetic studies have isolated four viable, spontaneous mouse mutants disrupted in growth, leading to dwarfism. Pygmy is unique among these mutants because its phenotype cannot be explained by aberrations in the growth hormone-insulin-like growth factor endocrine pathway. Here we show that the pygmy phenotype arises from the inactivation of Hmgi-c (ref. 6), a member of the Hmgi family which function as architectural factors in the nuclear scaffold and are critical in the assembly of stereospecific transcriptional complexes. Hmgi-c and another Hmgi family member, Hmgi(gamma) (ref. 10), were found to be expressed predominantly during embryogenesis. The HMGI proteins are known to be regulated by cell cycle-dependent phosphorylation which alters their DNA binding affinity. These results demonstrate the important role of HMGI proteins in mammalian growth and development.

Disruption of the architectural factor HMGI-C: DNA-binding AT hook motifs fused in lipomas to distinct transcriptional regulatory domains.

Lipomas are one of the most common mesenchymal neoplasms in humans. They are characterized by consistent cytogenetic aberrations involving chromosome 12 in bands q14-15. Interestingly, this region is also the site of rearrangement for other mesenchymally derived tumors. This study demonstrates that HMGI-C, an architectural factor that functions in transcriptional regulation, has been disrupted by rearrangement at the 12q14-15 chromosomal breakpoint in lipomas. Chimeric transcripts were isolated from two lipomas in which HMGI-C DNA-binding domains (AT hook motifs) are fused to either a LIM or an acidic transactivation domain. These results, identifying a gene rearranged in a benign neoplastic process that does not proceed to a malignancy, suggest a role for HMGI-C in adipogenesis and mesenchyme differentiation.

Ifg, Gli, Mdm1, Mdm2, and Mdm3: candidate genes for the mouse pg locus.

Various genes that mapped to the distal end of Chromosome (Chr) 10 were considered as possible candidates for the mouse pygmy (pg) locus. Probes derived from Ifg, Gli, Mdm1, Mdm2, and Mdm3 (Mdm2 and Mdm3 are genes that are coamplified with Mdm1 on the same double minute chromosomes in 3T3DM cells) were used for Southern analysis of DNA from wild-type mice and various pg mutants. In addition, the chromosomal locations of Ifg, Gli, Mdm1, Mdm2, and Mdm3 were determined by interspecific backcross analysis with progeny derived from matings of [(C57BL/6J x Mus spretus)F1 x C57BL/6J] mice. The mapping data indicate that the Mdm loci are linked to each other and to Ifg, pg, and Gli in the distal region of mouse Chr 10. Both the mapping data and the Southern analysis confirm that Mdm1, Mdm2, Mdm3, Ifg, and Gli are distinct from pg.