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Case Reports
. 2000 May 9;97(10):5214-9.
doi: 10.1073/pnas.090525897.

Nuclear structure in normal and Bloom syndrome cells

V Yankiwski  1 , R A Marciniak, L Guarente, N F Neff
Affiliations
Case Reports

Nuclear structure in normal and Bloom syndrome cells

V Yankiwski et al. Proc Natl Acad Sci U S A. .

Abstract

Bloom syndrome (BS) is a rare cancer-predisposing disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. BLM, the protein altered in BS, is a RecQ DNA helicase. This report shows that BLM is found in the nucleus of normal human cells in the nuclear domain 10 or promyelocytic leukemia nuclear bodies. These structures are punctate depots of proteins disrupted upon viral infection and in certain human malignancies. BLM is found primarily in nuclear domain 10 except during S phase when it colocalizes with the Werner syndrome gene product, WRN, in the nucleolus. BLM colocalizes with a select subset of telomeres in normal cells and with large telomeric clusters seen in simian virus 40-transformed normal fibroblasts. During S phase, BS cells expel micronuclei containing sites of DNA synthesis. BLM is likely to be part of a DNA surveillance mechanism operating during S phase.

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Figures

Figure 1
Figure 1
Localization of α-BLM and sites of DNA replication, repeated sequence elements, and ND10s. (A–C) Normal human diploid fibroblasts (HG2619 or WI38) double-labeled with α-BLM (α-rabbit TR/2°) and α-BrdUrd mouse (α-mouse FITC/2°). Representative BrdUrd patterns are shown: pattern 3, middle to late S phase (A); patterns 1 and 2, early S phase (B); pattern 4, late S phase (C); pattern 5, small numbers of foci (not shown). (D) HG2619 cell stained with α-BLM (α-rabbit FITC/2°) and α-nucleolar human autoimmune sera ANA-N (α-human TR/2°). (E) HG2619 cell stained with α-BLM (α-rabbit FITC/2°) and α-WRN (α-rabbit TR/2°). (F) HG2619 cells stained with α-BLM (α-rabbit FITC/2°) and α-centromere human autoimmune sera ANA-C (α-human TR/2°). (G) WI38 cell stained with α-BLM (α-rabbit TR/2°) and hybridization of a DIG-labeled telomeric sequence probe (α-DIG FITC/2°). (H) SV40-transformed-BS fibroblast cell (HG2522) stained with α-TRF1 rabbit (TR α rabbit/2°) and hybridization of a DIG-labeled telomeric sequence probe (α-DIG FITC/2°). (I) SV40-transformed normal fibroblast cell (HG2855) stained with α-BLM (α-rabbit TR/2°) and hybridization of a DIG-labeled telomeric sequence probe (α-DIG FITC/2°). (J) Confocal image of a normal cell (HG2619) stained with α-BLM (α-rabbit TR/2°) and α-PML (α-mouse FITC/2°). (K) WI38 cells stained with α-BLM (α-rabbit TR/2°), α-PML (α-mouse FITC/2°). (L) Confocal image of HG2619 cell stained with α-RPA (α-rabbit FITC/2°) and α-PML (α-mouse TR/2°). (M) BS cell (HG2940) stained with α-RPA (α-rabbit FITC/2°) and α-PML (α-mouse TR/2°). (N) BS cell (HG3002) stained with α-RPA (α-rabbit TR/2°) and α-BrdUrd (α-mouse FITC/2°). (O) BS cell (HG3005) stained with 4′,6-diamidino-2-phenylindole and hybridization of a DIG-labeled telomeric sequence probe (α-DIG FITC/2°). (P) BS cell (HG2940) stained with 4′,6-diamidino-2-phenylindole and α-ANA-C (α-human TR/2°).
Figure 2
Figure 2
Patterns of α-BLM staining during the cell cycle. Normal human diploid fibroblasts (HG2619) were serum-starved for 48 h and returned to growth by serum addition. Cells were harvested every hour and stained with α-BLM (α-rabbit TR/2°). Slides were pulse-labeled with BrdUrd and stained with α-BrdUrd (α-mouse FITC/2°). (A) Cells on slides were observed and counted for α-BLM staining and BrdUrd incorporation. (B) Distribution of α-BLM staining patterns as a function of time after serum addition. (C) Representative BLM patterns and a field of cells at 14 h after serum addition stained with α-BLM (α-rabbit TR/2°).
Figure 3
Figure 3
Analysis of telomere repeat lengths of genomic DNA from a consanguineous family of Ashkenazi Jewish decent. (A) Pedigree of family. Affected individuals are indicated by filled symbols. The age of the person when the sample was drawn is indicated. (B) Whole blood DNA from eight sibs ranging in ages from 2 to 19 years and the paternal grandmother (62 years of age) was digested with restriction endonucleases and displayed. A lanes are genomic DNA digests with AluI restriction enzyme. H lanes are genomic DNA digests with restriction enzyme HinfI. Data are shown in Table 2.
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