Delayed replication timing leads to delayed mitotic chromosome condensation and chromosomal instability of chromosome translocations - PubMed (original) (raw)

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

L Smith et al. Proc Natl Acad Sci U S A. 2001.

Abstract

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.

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Figures

Figure 1

Chromosome 3q translocations. (A) Karyotypic and FISH analysis of the i(3q), the t(3q;9q), and the der(3q) chromosomes. The i(3q) was isolated in microcell hybrids from the rhabdomyosarcoma cell line RH30 and the t(3;9) and der(3q) chromosomes were from the SCLC line CRL-5845. Mitotic spreads were analyzed by G-banding (G) followed by FISH with a chromosome 3 paint (3); or by FISH with an ATR probe followed by FISH with a chromosome 3 paint (3). Mitotic chromosome spreads from cells containing the i(3q) (B), the der(3q) (C), or the t(3q;9q) (D). The chromosome 3 translocations were identified by using either a chromosome 3-specific alpha satellite probe (B) or a human-specific pancentromeric probe (C and D). Arrows indicate the chromosome 3 translocations. The DNA was visualized by staining with either DAPI (B) or propidium iodide (A, C, and D). (E) The frequency of undercondensed chromosomes was determined in C2C12 microcell hybrids containing the i(3q), the t(3;9), the der(3q), or a nonrearranged chromosome 3. Metaphase spreads were prepared in the presence or absence of a 30-min colcemid treatment. A minimum of 100 spreads from the translocation lines, and 1,000 spreads from the nonrearranged chromosome 3 line were scored for undercondensed chromosomes.

Figure 2

Delay of mitotic phosphorylation of histone H3. Mitotic chromosome spreads containing the i(3q) were stained with an antibody specific for phosphorylation of serine 10 of histone H3 (B, D, and F). The i(3q)s in each spread were identified by using FISH with a chromosome 3 alpha satellite probe, and the chromosomes were visualized by using DAPI (A, C, and E). Two mitotic spreads with undercondensed chromosomes (A_–_D) and a mitotic spread with two i(3q) chromosomes with apparently normal condensation and phospho-H3 staining (E and F) are shown.

Figure 3

Delay of mitotic chromosome condensation in tumor cell lines. (A) Mitotic chromosome spreads were scored for the presence of chromosomes with DMC in tumor derived cells. Chromosomes with DMC were scored in mitotic spreads harvested either in the presence (+) or absence (−) of colcemid from the blood of two normal individuals (46XY and 46XX) and from the tumor-derived cell lines RH30, CRL-5845, CRL-5824, HTB-118, WERI-RB1, and HTB-81. Following propidium iodine staining, a minimum of 100 mitotic spreads was scored for each sample. Sequential G-banding (C) and FISH (B) with a chromosome 3 paint on a mitotic spread from RH30 cells prepared in the absence of colcemid. This probe hybridized to a nonrearranged chromosome 3 (arrow), and to a chromosome with DMC (✻ and arrow). (D and E) Mitotic chromosome spreads showing two copies of the der(1q) (D) that hybridize to a chromosome 1 paint (E). (F) Mitotic spreads were analyzed sequentially by G-banding (G) followed by FISH with a chromosome 1 paint (1). The DNA was visualized in the FISH analyses by staining with propidium iodide.

Figure 4

Delay of replication timing on chromosomes with DMC. (A) General scheme for replication timing experiments, showing the time of the BUDR pulse and the time of mitotic cell harvest for early and late replication. Incorporated BUDR was detected by using an FITC labeled anti-BUDR antibody, and the DNA was detected by using PI. (B) A representative mitotic spread from cells containing the i(3q) harvested for early replication. The arrows mark the chromosomes with DMC (B_–_D). (C) A representative mitotic spread from cells containing the i(3q) harvested for late replication. (D) A mitotic spread from cells containing the i(3q) harvested for late replication showing a banded pattern of BUDR incorporation. (E) A representative mitotic spread from the parental C2C12 cells harvested for early replication. The arrow marks a late replicating X chromosome. (F) A representative mitotic spread from C2(3n)-1 cells harvested for early replication. The arrow marks the nonrearranged human chromosome 3. (G and H) A representative mitotic spread from CRL-5845 harvested for late replication (G shows staining with PI only, and H shows PI plus BUDR).

Figure 5

Chromosomal instability. Numerous secondary alterations of the chromosome 3 translocations were observed in late passage (–20) cultures. Mitotic spreads were subjected to FISH analysis with the chromosome 3 painting probe. (A) A representative mitotic spread from late passage cultures of cells with the der(3q). The arrows mark numerous translocations involving human chromosome 3 and the C2C12 mouse chromosomes. (B) A mitotic spread from cells with the i(3q) with a radial chromosome involving the i(3q) and a mouse chromosome.

Figure 6

Models for DRT and chromosomal instability. (A) We are considering two possible mechanisms that could result in DRT/DMC. First, because all of the translocations with DRT/DMC involve deletion and/or rearrangements of one arm of the affected chromosome, it is possible that deletion or mutation of a cis element (shown in yellow) that normally establishes early replication timing has occurred. Deletion of this element would then result in delayed replication of the entire chromosome. Second, because all of the translocations with DRT/DMC involve translocations or rearrangements in or near the centromeres of the affected chromosomes, it is possible that this type of chromosomal rearrangement actively interferes with normal chromosome replication timing by some unknown mechanism. (B) Schematic diagram of: DNA, DNA replication, chromatid condensation, and chromatid separation of a chromosome with DRT/DMC. We propose that delayed replication results in incomplete replication and/or incomplete mitotic chromosome condensation that persists into mitosis. Consequently, during chromatid separation either unreplicated DNA causes a break, or incomplete condensation results in a “weak” spot that causes a break during chromatid separation at anaphase. This model was adapted from the “late-replicating DNA” model for fragile site expression (21).

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Delayed replication timing leads to delayed mitotic chromosome condensation and chromosomal instability of chromosome translocations - PubMed (original) (raw)