Genomic lesions induced by DNA replication stress

Given the conservation of CFS in the mouse and human genomes (Glover, Berger et al. 1984; Helmrich, Stout-Weider et al. 2006; Smith, Zhu et al. 2006) it appears that some of the CNCs identified in the induced stem cells map to CFS. Specifically, the deletions in the Rora and Ptprg genes map to the human CFS FRA15A and FRA3B, respectively. Several of the remaining identified CNCs targeted large genes, again suggesting DNA replication stress as the culprit (Helmrich, Stout-Weider et al. 2006;

Smith, Zhu et al. 2006). To explore the spectrum of CNCs generated by DNA replication stress, we treated cells for 4 weeks with low doses of aphidicolin, a prototypical agent for inducing DNA replication stress, and then monitored CNCs by cGH (Figure 2.9a). For this experiment, we employed a mouse–human hybrid cell

line, GM11713A, which contains a single copy of human chromosome 3, thus, facilitating the detection of deletions. This cell line was previously used to map aphidicolin-induced deletions within the FHIT gene (Durkin, Ragland et al. 2008).

Six aphidicolin-treated clones were isolated for cGH analysis spanning the entire chromosome 3. Two clones were essentially identical, except for one small deletion, suggesting that they arose from a common ancestor cell. The concordance of the findings for these two clones provided a validation of the quality of the cGH analysis, but reduced the number of independent clones from six to five. In addition to deletions targeting the FHIT gene in FRA3B in four out of the five clones, two clones had deletions within the PTPRG gene, also mapping to the FRA3B CFS (Figure 2.9b). Deletions were further found in the genes dedicator of cytokinesis 3 (DOCK3), cell adhesion molecule 2 (CADM2), limbic system-associated membrane protein (LSAMP) and N-acetylated alpha-linked acidic dipeptidase-like 2 (NAALADL2), none of which map within established CFS (Figure 2.10). Finally, a

gain was detected within the ELKS–RAB6-interacting/CAST family member 2 (ERC2) gene (Figure 2.9b). An interesting feature of this analysis is that all the identified CNCs mapped to large genes. In fact, the CNCs targeted 7 of the 17 largest genes of chromosome 3, whereas no CNCs were detected in any of the remaining annotated genes of chromosome 3 (Figure 2.9c). Further, while DNA replication stress is usually associated with deletions, one of the observed CNCs was a gain.

Similar findings have been obtained by another group using a similar experimental approach (Durkin, Ragland et al. 2008; Arlt, Mulle et al. 2009). Thus, based on the spectrum of CNCs induced by aphidicolin, we propose that of the eight CNCs observed in the induced stem cells (mammary stem cells and iPS cells), six may be linked to DNA replication stress.

Figure 2.1 Reprogramming of progenitor mammary cells to stem cells by MycER

(a and b) PKH26-negative cells, which are devoid of stem cells, were isolated from secondary mammospheres and infected with a lentivirus-expressing MycER or with a control virus (Empty Vector). The cells were cultured under non-adherent conditions and mammosphere number (a) and cumulative cell number (b) were determined at each passage. Results are presented as means±1 S.D.

(c) Representative images of the cultures at passages 1 and 2.

(d) Repopulation of a cleared mouse fat pad by MycER-expressing mammospheres originally prepared from PKH26-negative cells with no

evidence of tumor development

Figure 2.2 Stem cell reprogramming of primary mammary cells by MycER (a) Primary mammary cells prepared from wild-type or p53-/- mice were infected with a lentivirus-expressing MycER or with a control virus (Empty Vector) and cultured under non-adherent conditions. In some cultures TAM was added to induce high levels of MycER activity. The number of mammospheres and the cumulative number of cells were determined at each passage. Results are presented as means±1 S.D.

(b) Repopulation of a cleared mouse fat pad by MycER-expressing mammospheres prepared by lentiviral infection of primary mammary cells. There are no signs of tumor development in the replanted mammary gland

Figure 2.3 High levels of MycER protein induce p53 activation and apoptosis

(a) Expression of MycER, as ascertained by (IF), in disaggregated mammosphere cells cultured in the absence and presence of 4-hydroxytamoxifen (TAM). The mammospheres were derived from primary cells infected 3 days earlier with a control lentivirus (Empty Vector) or a lentivirus expressing MycER. For quantitation, the fluorescent signal corresponding to the nucleus of between 20-30 cells for each condition was measured using ImageJ and shown as mean + one standard deviation.

(b) High levels of MycER activity induce activation of p53 and apoptosis. 4-hydroxytamoxifen (TAM) was added to mammosphere cultures of infected primary mammary cells, either as a single addition on day 1 after infection (+s) or daily for 7 days (+d). After 7 days the cells were harvested and analysed by immunoblotting. For activation of p53 in response to DNA damage, cells from primary mammospheres were treated with 2 µg/ml adriamycin for 8 h. p53-pS15, p53 phosphorylated on Ser15. Vinculin serves as a standard.

Figure 2.4 Genomic instability in MycER-induced mammary stem cells

(a) Protocol for isolation of genomic DNA from wild-type mammary cells and from MycER-reprogrammed stem cell clones.

(b) cGH analysis of four MycER-reprogrammed stem cell clones. Selected regions of mouse chromosomes 9, 13 and 14 are indicated. The results are expressed as log2 ratios of the intensity signals of the stem cell clone DNA to the control DNA. Each black bar represents the average of 10 probes with 1388-bp median spacing. The red lines represent the statistical average and show discontinuities, when a series of averaged probe data deviates in a statistically significant manner from its neighbors.

Transcripts are indicated by blue lines and the names of select genes are indicated.

Figure 2.5 Characterization of iPS cells induced by 3 factors (OSK)

(a) Morphology of iPS-OSK clone #1 on MEF feeder layers. Phase-contrast and fluorescence images depicting the morphology of the colonies and expression of Oct4- GFP, respectively, are shown.

(b) Expression of pluripotency genes in iPS-OSK cells. Expression levels of Nanog, Oct4 and Sox2 were determined by quantitative PCR for iPS-OSK clones #1 and #2, for three different cultures of MEFs and for embryonic stem (ES) cells. Means and standard deviations from three different experiments are shown. Expression levels in the ES cells were set to 100 (arbitrary expression units).

(c) Differentiation of iPS-OSK clone #1 in vitro. Phase-contrast images and fluorescence images to monitor embryoid body formation and Oct4-GFP expression, respectively, were acquired over a period of 10 days after shifting the iPS cells to tissue culture plates without feeder layers and removing LIF from the media.

Figure 2.6 Genomic instability in iPS cells induced by OSK

(a) Protocol for isolation of genomic DNA from iPS cells and from non-infected MEFs.

(b) cGH analysis of six iPS cell clones. Two regions of mouse chromosome 8 are indicated. The results are expressed as log2 ratios of the intensity signals of the iPS cell DNA to the control DNA.

Each black bar represents a single probe with 1388-bp median spacing. The red lines represent the statistical average and show discontinuities, when a series of averaged probe data deviates in a statistically significant manner from its neighbors. Transcripts are indicated by blue lines and the names of select genes are indicated.

Figure 2.7 Characterization of iPS cells induced by four factors (OSKC) (a) Morphology of iPS-OSKC clones 1 and 2 by phase-contrast microscopy.

(b) Staining of iPSOSKCclones 1 and 2 for alkaline phosphatase activity.

(c) Expression of pluripotency markers in iPS-OSKC cells. Expression levels of SSEA-1 and Oct4-GFP in iPS-OSK clone 1 and 2 cells and in the MEFs from which these iPS cells were derived, as determined by flow cytometry. The percentages of cells expressing high or low levels of SSEA-1 and Oct4-GFP are indicated by purple-colored numbers.

(d) Expression of pluripotency genes in iPS-OSKC cells. Levels of Oct4, Zfp296, Eras and Fgf4 in iPS-OSKC clones 1 and 2, in embryonic stem (ES) cells and in MEFs were determined by PCR. Nat1 expression serves as a standard.

(e) Morphology of embryoid bodies formed after differentiation of iPS-OSKC clones 1 and 2 for 7 days in vitro, as revealed by hematoxylin–eosin staining

Figure 2.8 Genomic instability in iPS cells induced by OSKC

(a) Protocol for isolation of genomic DNA from iPS cells and from non-infected MEFs.

(b) cGH analysis of two iPS cell clones. The array hybridization for clone 1 was performed twice and both replicates are shown. Selected regions of mouse chromosomes 7 and 8 are indicated. The results are expressed as log2 ratios of the intensity signals of the iPS cell DNA to the control DNA. Each black bar represents the average of 10 probes with 1388-bp median spacing. The red lines represent the statistical average and show discontinuities, when a series of averaged probe data deviates in a statistically significant manner from its neighbours. Transcripts are indicated by blue lines and the names of select genes are indicated.

Figure 2.9 Genomic instability induced by chronic DNA replication stress

(a) Protocol for isolation of genomic DNA from aphidicolin-treated GM11713A cells and untreated cells.

(b) cGH analysis of six aphidicolin-treated clones. Selected regions of human chromosome 3 are indicated. The results are expressed as log2 ratios of the intensity signals of the aphidicolin-treated cell DNA to the control DNA. Each black bar represents the average of 10 probes with 475-bp median spacing. The red lines represent the statistical average and show discontinuities, when a series of averaged probe data deviates in a statistically significant manner from its neighbors. Transcripts are indicated by blue lines and the names of select genes are indicated. The fifth and six clones are related to each other and are referred to as subclones 5a and 5b.

(c) List of the 17 largest genes of human chromosome 3, according to their size, indicating also the presence of identified genomic aberrations in the five aphidicolin-treated clones. The status column indicates the number of clones with a genomic aberration and the type of aberration. del, deletion

Figure 2.10 Genomic instability induced by chronic DNA replication stress.

Comparative genomic hybridization analysis of the aphidicolin-treated clones shown in Figure 2.7 focusing on additional selected regions of human chromosome 3. The results are expressed as log2 ratios of the intensity signals of the aphidicolin-treated cell DNA to the control DNA. Each black bar represents the average of 10 probes with 475 bp median spacing. The red lines represent the statistical average and show discontinuities, when a series of averaged probe data deviates in a statistically significant manner from its neighbors. Transcripts are indicated by blue lines and the names of select genes are indicated. The fifth and six clones are related to each other and are referred to as subclones 5a and 5b.

CHAPTER 3