Leslie Smith

Frequent aneuploidy among normal human hepatocytes.

Murine hepatocytes become polyploid and then undergo ploidy reversal and become aneuploid in a dynamic process called the ploidy conveyor. Although polyploidization occurs in some types of human cells, the degree of aneuploidy in human hepatocytes is not known. We isolated hepatocytes derived from healthy human liver samples and determined chromosome number and identity using traditional karyotyping and fluorescence in situ hybridization. Similar to murine hepatocytes, human hepatocytes are highly aneuploid. Moreover, imaging studies revealed multipolar spindles and chromosome segregation defects in dividing human hepatocytes. Aneuploidy therefore does not necessarily predispose liver cells to transformation but might promote genetic diversity among hepatocytes.

Localization of the fibrillin (FBN) gene to chromosome 15, band q21.1

Fibrillin (FBN), a large extracellular matrix glycoprotein, is an important component of structures called microfibrils. Because fibrillin microfibrils appear to be abnormal in patients with the Marfan syndrome, fibrillin is a candidate for the gene defect in the Marfan syndrome. Derived clones from fibrillin cDNA were used as probes in isotopic and nonisotopic in situ hybridization studies to map the chromosomal location of the fibrillin gene. Fluorescent signals were found on chromosome 15 band q21.1; an excess of silver grains was noted over a similar region of chromosome 15 following in situ hybridization with a tritium-labeled probe. These results are consistent with linkage studies that localize the Marfan gene to chromosome 15.

The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts.

Sequence-dependent formation and lack of repair of polycyclic aromatic hydrocarbon-induced DNA adducts correlates well with the positions of p53 mutational hotspots in smoking-related lung cancers (, ). The mycotoxin aflatoxin B1 (AFB1) is considered to be a major causative agent in hepatocellular carcinoma (HCC) in regions with presumed high food contamination by AFB1. A unique mutational hotspot, a G to T transversion at the third base of codon 249 of the p53 gene is observed in these tumors. To test whether a selectivity of AFB1 adduct formation is related to this peculiar mutational spectrum, we have mapped AFB1-DNA adducts at nucleotide resolution using ligation-mediated PCR and terminal transferase-dependent PCR. Human HepG2 cells were exposed to AFB1 metabolically activated in the presence of rat liver microsomes. Significant adduct formation was seen at the third base of codon 249. However, this was not the major site of AFB1 adducts and strong adduction was also observed at codons 226, 243, 244, 245 and 248 in exon 7 of the p53 gene and at several codons in exon 8. The damage at codon 249 does not consist of a unique abasic site or ring-opened aflatoxin B1 adduct but rather is consistent with the principal N7-guanine adduct of AFB1. Time course experiments indicate that, under the conditions used, AFB1 adducts are not removed in a strand-selective manner and adduct removal from the third base of codon 249 proceeds at a relatively fast rate (50% in 7 h). The incomplete correspondence between sites of persistent AFB1 damage and the specific codon 249 mutation suggests that AFB1 may not be involved in mutation of this site or that additional mechanisms such as parallel infection with hepatitis B virus may be required for selection of codon 249 mutants in HCC.

Duplication of ATR inhibits MyoD, induces aneuploidy and eliminates radiation-induced G1 arrest.

Chromosome 3q alterations occur frequently in many types of tumours. In a genetic screen for loci present in rhabdomyosarcomas, we identified an isochromosome 3q [i(3q)], which inhibits muscle differentiation when transferred into myoblasts. The i(3q) inhibits MyoD function, resulting in a non-differentiating phenotype. Furthermore, the i(3q) induces a ‘cut’ phenotype, abnormal centrosome amplification, aneuploidy and loss of G1 arrest following γ-irradiation. Testing candidate genes within this region reveals that forced expression of ataxiatelangiectasia and rad3-related (ATR) results in a phenocopy of the i(3q). Thus, genetic alteration of ATR leads to loss of differentiation as well as cell-cycle abnormalities.

Mapping of the ACTH, MSH, and neural (MC3 and MC4) melanocortin receptors in the mouse and human

The melanocortin peptides regulate a wide variety of physiological processes, including pigmentation and glucocorticoid production, and also have several activities in the central and peripheral nervous systems. The melanocortin receptor family includes the melanocytestimulating hormone receptor (MSH-R), adrenocorticotropic hormone receptor (ACTH-R), and two neural receptors, MC3-R and MC4-R. In the human these receptors map to 16q24 (MSH-R), 18p11.2 (ACTH-R), 20q13.2 (MC3-R), and 18q22 (MC4-R). The corresponding locations in the mouse are 8, 18, and 2; a variant for mapping MC4-R has not yet been identified. The data reported here also show that the neural MC3 receptor maps close to a disease locus for benign neonatal epilepsy in human and near the El-2 epilepsy susceptibility locus in the mouse.

Delayed replication timing leads to delayed mitotic chromosome condensation and chromosomal instability of chromosome translocations

Chromosomal rearrangements are found in virtually all types of human cancers. We show that certain chromosome translocations display a delay in mitotic chromosome condensation that is associated with a delay in the mitosis-specific phosphorylation of histone H3. This delay in mitotic condensation is preceded by a delay in both the initiation as well as the completion of chromosome replication. In addition, chromosomes with this phenotype participate in numerous secondary translocations and rearrangements. Chromosomes with this phenotype were detected in five of seven tumor-derived cell lines and in five of thirteen primary tumor samples. These data suggest that certain chromosomal rearrangements found in tumor cells cause a significant delay in replication timing of the entire chromosome that subsequently results in delayed mitotic chromosome condensation and ultimately in chromosomal instability.

Amplification of MDM2 inhibits MyoD-mediated myogenesis.

One obvious phenotype of tumor cells is the lack of terminal differentiation. We previously classified rhabdomyosarcoma cell lines as having either a recessive or a dominant nondifferentiating phenotype. To study the genetic basis of the dominant nondifferentiating phenotype, we utilized microcell fusion to transfer chromosomes from rhabdomyosarcoma cells into C2C12 myoblasts. Transfer of a derivative chromosome 14 inhibits differentiation. The derivative chromosome 14 contains a DNA amplification. MDM2 is amplified and overexpressed in these nondifferentiating hybrids and in the parental rhabdomyosarcoma. Forced expression of MDM2 inhibits MyoD-dependent transcription. Expression of antisense MDM2 restores MyoD-dependent transcriptional activity. We conclude that amplification and overexpression of MDM2 inhibit MyoD function, resulting in a dominant nondifferentiating phenotype.

Chromosomes with delayed replication timing lead to checkpoint activation, delayed recruitment of Aurora B and chromosome instability

Certain chromosome rearrangements display a significant delay in chromosome replication timing (DRT) that is associated with a subsequent delay in mitotic chromosome condensation (DMC). DRT/DMC chromosomes are common in tumor cells in vitro and in vivo and occur frequently in cells exposed to ionizing radiation. A hallmark for these chromosomes is the delayed phosphorylation of serine 10 of histone H3 during mitosis. The chromosome passenger complex, consisting of multiple proteins including Aurora B kinase and INCENP is thought to be responsible for H3 phosphorylation, chromosome condensation and the subsequent segregation of chromosomes. In this report, we show that chromosomes with DRT/DMC contain phosphorylated Chk1, consistent with activation of the S–M phase checkpoint. Furthermore, we show that INCENP is recruited to the DRT/DMC chromosomes during all phases of mitosis. In contrast, Aurora B kinase is absent on DRT/DMC chromosomes when these chromosomes lack serine 10 phosphorylation of H3. We also show that mitotic arrest deficient 2 (Mad2), a member of the spindle assembly checkpoint, is present on DRT/DMC chromosomes at a time when the normally condensed chromosomes show no Mad2 staining, indicating that DRT/DMC activates the spindle assembly checkpoint. Finally, cells with DRT/DMC chromosomes have centrosome amplification, abnormal spindle assembly, endoreduplication and significant chromosome instability.

Intrachromosomal location of the telomeric repeat (TTAGGG)n

Eukaryotic telomeres are specialized DNA-protein structures that are thought to ensure chromosomal stability and complete replication of the chromosome ends. All telomeres which have been studied consist of a tandem array of G-rich repeats which seem to be sufficient for telomere function. Originally, the human telomeric repeat (TTAGGG)n was assumed to be exclusively located at the very end of all human chromosomes. More recent evidence, however, suggests an extension into proterminal regions. Very little is known about the interstitial distribution of telomeric repeats. Here we present evidence for the presence of (TTAGGG)n repeats in internal loci on the long and short arms of different human chromosomes. In addition, we studied the genomic organization of these repeats in more detail and discuss possible functions of interstitial telomeric repeats in the human genome.

An autosomal locus that controls chromosome-wide replication timing and mono-allelic expression

Mammalian DNA replication initiates at multiple sites along chromosomes at different times, following a temporal replication program. Homologous alleles typically replicate synchronously; however, mono-allelically expressed genes such as imprinted genes, allelically excluded genes and genes on the female X chromosome replicate asynchronously. We have used a chromosome engineering strategy to identify a human autosomal locus that controls this replication timing program in cis. We show that Cre/loxP-mediated rearrangements at a discrete locus at 6q16.1 result in delayed replication of the entire chromosome. This locus displays asynchronous replication timing that is coordinated with other mono-allelically expressed genes on chromosome 6. Characterization of this locus revealed mono-allelic expression of a large intergenic non-coding RNA, which we have named asynchronous replication and autosomal RNA on chromosome 6, ASAR6. Finally, disruption of this locus results in the activation of the previously silent alleles of linked mono-allelically expressed genes. We previously found that chromosome rearrangements involving eight different autosomes display delayed replication timing, and that cells containing chromosomes with delayed replication timing have a 30-80-fold increase in the rate at which new gross chromosomal rearrangements occurred. Taken together, these observations indicate that human autosomes contain discrete cis-acting loci that control chromosome-wide replication timing, mono-allelic expression and the stability of entire chromosomes.