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. 2009 Mar;11(3):260–268. doi: 10.1593/neo.81384

Recurrent RECQL4 Imbalance and Increased Gene Expression Levels Are Associated with Structural Chromosomal Instability in Sporadic Osteosarcoma1,2

Georges Maire *, Maisa Yoshimoto *, Susan Chilton-MacNeill †, Paul S Thorner ‡, Maria Zielenska †,‡, Jeremy A Squire *
PMCID: PMC2647728  PMID: 19242607

Abstract

Osteosarcoma (OS) is an aggressive bone tumor with complex abnormal karyotypes and a highly unstable genome, exhibiting both numerical- and structural-chromosomal instability (N- and S-CIN). Chromosomal rearrangements and genomic imbalances affecting 8q24 are frequent in OS. RECQL4 gene maps to this cytoband and encodes a putative helicase involved in the fidelity of DNA replication and repair. This protective genomic function of the protein is relevant because often patients with Rothmund-Thomson syndrome have constitutional mutations of RECQL4 and carry a very high risk of developing OS. To determine the relative level of expression of RECQL4 in OS, 18 sporadic tumors were studied by reverse transcription-polymerase chain reaction. All tumors overexpressed RECQL4 in comparison to control osteoblasts, and fluorescence in situ hybridization analysis of tumor DNA showed that expression levels were strongly copy number-dependent. Relative N- and S-CIN levels were determined by classifying copy number transitions within array comparative genomic hybridization profiles and by enumerating the frequency of break-apart fluorescence in situ hybridization within 8q24 using region-specific and control probes. Although there was no evidence that disruption of 8q24 in OS led to an elevated expression of RECQL4, there was a marked association between increased overall levels of S-CIN, determined by copy number transition frequency and higher levels of RECQL4.

Introduction

Osteosarcoma (OS) is the most common primary bone malignancy and is characterized by complex chromosomal abnormalities that vary widely from cell to cell. These tumors exhibit a high degree of aneuploidy, gene amplification, and multiple unbalanced chromosomal rearrangements. A combined approach of molecular cytogenetics techniques [comparative genomic hybridization (CGH), spectral karyotyping, or multicolor banding] together with classic G-banded cytogenetics analysis of OS tumors describe complex karyotypes with multiple numerical and structural chromosomal aberrations. Collectively, these studies [1–12] have highlighted the unique and highly unstable karyotype of OS.

Two distinct processes governing genome stability may be disrupted in cancer cells: those that affect numerical segregation and ploidy of chromosomes and those that affect the fidelity of DNA replication/repair and lead to structural chromosome aberrations (reviewed in [13]). The complexity of the OS genome likely arises as a consequence of chromosomal instability (CIN), generated by both numerical and structural chromosome abnormalities. Numerical patterns of chromosomal aberration have been referred to as N-CIN, whereas CIN that leads to elevated levels of structural change has been termed S-CIN. The development of high-resolution array comparative genomic hybridization (aCGH) methods provides an opportunity to analyze genomic complexity at both the N-CIN and S-CIN level using whole-genome imbalance plots. In this approach, the respective distributions of copy number alteration present within the entire aCGH profile can be used to determine the relative contributions of S-CIN [as defined by copy number transitions (CNTs) [14], within each chromosome] or N-CIN (as defined by chromosomal imbalance affecting an entire chromosome) to the overall complexity of the OS genome in a given tumor. Previously [15], we have used aCGH analysis of OS to map chromosomal regions recurrently subject to genomic changes such as gene amplification. In the current article, we further interrogate aCGH profiles to measure levels of S-CIN and N-CIN in OS in the context of genomic and molecular changes of cytoband 8q24.

Some chromosomal regions are more frequently involved in genomic aberrations in OS, namely, 1p35-p36, 6p12-p21, 8q23-q24, 17p11-p12, and 19p13 [4] (reviewed in [16]). The 8q24 region is of particular interest to OS because a number of genes both directly and indirectly implicated in OS oncogenesis map to this region. The MYC oncogene at 8q24 is highly amplified in a subset of OS [17,18], is overexpressed more frequently in relapsed and metastatic OS [19], and is often amplified in a wide variety of carcinomas [20]. RECQL4 maps to this same cytoband and the gene encodes a putative helicase involved in the fidelity of DNA replication, and this protective genomic function of the protein is provocative because patients with constitutional mutations of RECQL4 have Rothmund-Thomson syndrome (RTS) and carry a very high risk of developing an OS [21]. Approximately two-thirds of patients with a clinical diagnosis of RTS will have RECQL4 mutations. The other one third likely represents genetic heterogeneity and have mutations in another gene(s). The RTS patients with RECQL4 mutations have a much higher risk of developing OS compared with the RTS patients without RECQL4 mutations [22]. Moreover, the 8q24 cytoband is now of particular interest to cancer biologists because recent genome-wide association studies have identified multiple neighboring regions within a 600-kb segment of chromosome 8q24 that harbors variants associated with predisposition to prostate, colon, and bladder cancers [23–25]. The most likely candidate gene within the 8q24 region that could contribute directly to the CIN phenotype of OS is the RECQL4 gene, which encodes a helicase member of the RecQ family [26].

In this study, we address the hypothesis that deregulation of RECQL4 expression, caused by the 8q24 rearrangements, could underlie the high rate of CIN observed in OS. Complex genomic alterations and amplifications at 8q24 were of particular interest because the affected regions are relatively small; they have been found to be aberrant in multiple OS samples and they are located in a region of the human genome strongly implicated in tumorigenesis and DNA repair. If genomic alterations occur near the RECQL4 region, it is conceivable that such changes lead to deregulation of the locus, and this may compromise its repair and DNA maintenance functions, with consequences for the entire genome's integrity [26–30]. In the present study, we used 18 OS tumor samples to investigate whether RECQL4 gene expression levels were linked to the extent and type of CIN (N-CIN and S-CIN) throughout the OS genome and specifically at 8q24.

Materials and Methods

Patient Tumors

The collection of frozen tissue specimens (n = 18), archival formalinfixed, paraffin-embedded OS sections (n = 12), and clinicopathologic data was obtained and handled in accordance with the Hospital for Sick Children Research Ethics guideline (Toronto, Canada). The OS specimens and corresponding paraffin-embedded specimens consisted of resected or biopsy tumor tissue obtained at diagnosis. Hematoxylin and eosin-stained sample sections were subjected to standard histopathologic evaluation to determine the tumor content and the pathologic grade according to the World Health Organization [31]. All samples presented a tumor content higher than 90%. The clinical and histologic features of presented OS cohort are detailed in Table 1.

Table 1.

Clinical Data of the 18 OSs.

No. Sex Age (years) Localization Sample Subtype Grade
OS1 F 9 Tibia Biopsy Osteoblastic High
OS2 M 14 Femur Lesion Osteoblastic High
OS3 M 13 Femur Biopsy Osteoblastic Intermediate
OS4 M 7 Humerus Biopsy Poorly differentiated High
OS5 F 12 Femur Biopsy Poorly differentiated High
OS6 M 14 Femur Biopsy Osteoblastic High
OS7 M 7 Femur Biopsy Osteoblastic High
OS8 M 13 Femur Resection Osteoblastic High
OS9 F 13 Humerus Biopsy Osteoblastic High
OS10 M 8 Tibia Resection Osteoblastic High
OS11 M 13 Femur Biopsy Osteoblastic High
OS12 M 17 Lung Resection Osteoblastic High
OS13 M 9 Leg Biopsy Poorly differentiated High
OS14 F 12 Femur Biopsy Osteoblastic Intermediate
OS15 F 14 Femur Biopsy Osteoblastic High
OS16 F 17 Tibia Resection Osteoblastic High
OS17 M 15 Tibia Resection Osteoblastic High
OS18 M 15 Femur Biopsy Poorly differentiated High
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F indicates female; M, male.

Cell Culture

The RTS primary fibroblasts were obtained from the Corriell Institute for Medical Research (AG18371; Camden, NJ) [32]. These fibroblasts do not express RECQL4 because of an 11-bp intronic deletion, which disrupts the splicing and compromises its expression [32,33]. They were cultured in alpha-minimum essential medium (Invitrogen, Burlington, Ontario, Canada) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen). Osteoblasts were obtained from PromoCell (Heidelberg, Germany) and cultured in osteoblast culture medium (PromoCell).

RNA Extraction and Semiquantitative Reverse Transcription-Polymerase Chain Reaction

The mRNA level of RECQL4 was examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), using the housekeeping gene PBGD as a calibration control. Total RNA from snap-frozen OS tumors (18 samples) and from cultured cells (RTS fibroblasts and osteoblasts) were isolated using the TRIzol Reagent (Invitrogen). The RNA quality was assessed by BioAnalyzer RNA 600 Nano Kit (Agilent Technologies, Palo Alto, CA). Total RNA from kidney and testis (Ambion, Foster City, CA) were used as positive control detection for low and high RECQL4 expressions, respectively [34]. Total RNA from RTS fibroblasts were used as negative control [33,35]. Coamplification of RECQL4 and PBGD genes was performed on 150 ng of total RNA by applying the one-step RT-PCR method (Superscript One-Step RT-PCR III; Invitrogen) according to the manufacturer's instructions. RECQL4 forward (5′-CTCATCTAAGGCATCCACCC-3′) and reverse (5′-CTGTGACATCGCTGTAACCA-3′) primers were designed to amplify a 188-bp fragment (Accession Number NM_004260). PBGD primers were designed to amplify a 127-bp fragment [36]. Primers for RECQL4 and PBGD were combined as follows: 0.2 µM of each forward and reverse RECQL4 primers and 0.15 µM of each forward and reverse PBGD primers. The reverse transcription and amplification conditions were performed using the MJ Research PTC200 thermocycler and consisted of an initial reverse transcription reaction at 57°C for 30 minutes, followed by denaturation (2 minutes at 94°C) and 30 cycles of amplification (94°C for 30 seconds; 57°C for 30 seconds; 68°C for 30 seconds). The final elongation reaction was performed at 68°C for 10 minutes. Genomic DNA contamination in every sample was excluded by omitting the reverse transcriptase in the RT-PCR.

The measurement and quantification of the 188-bp (RECQL4) and 127-bp (PBGD) coamplified fragments were performed using the DNA 1000 LabChip Kit (Agilent 2100 Bioanalyzer; Agilent Technologies) and the 2100 Expert Software (Agilent Technologies), respectively. PBGD was used for calibration, and the mRNA fold change of RECQL4 in OS cohort was compared with the normal osteoblasts. The SD from triplicate RT-PCR experiments was calculated for each sample.

Genome-wide Analysis of Chromosomal Instability

Ten OS samples for which sufficient total DNA could be extracted were hybridized against Human Genome CGH 44k microarrays (Agilent Technologies), spanning the entire human genome at a median resolution of 75 kb as described previously [15]. These aCGH data files have also been used to map the distribution of recurrently deleted and amplified regions in OS [15] and they are deposited in National Center for Biotechnology Information's Gene Expression Omnibus (GEO) Web site (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through the GEO Series accession number GSE9654. For each tumor, the normalized data were run through the Circular Binary Segmentation (CBS) algorithm [37], with a threshold of 0.5 (CGH Analytics, 3.5.14; Agilent Technologies). The CBS algorithm provides a list of aberrations (imbalanced genomic region with a unique abnormal CGH ratio), each determined by two CNTs, as defined by Ferreira et al. [14]. The distribution along the chromosome of these CNTs for each aberration was used to determine the respective contribution of N-CIN and S-CIN (Table W1). Briefly, an aberration with the starting and ending CNTs positioned within an arm of a chromosome was considered to be caused by copy number change arising from an unbalanced structural alteration and was scored as S-CIN. An aberration, which CNTs were positioned in telomeric or centromeric regions, was considered to result from copy number change affecting an entire chromosome or chromosome arm and was scored as N-CIN. All the aberrations were classified as N- or S-CIN-related and scored for each of the 10 OS tumors studied by aCGH. The aberrations on X and Y chromosomes were excluded from the analysis to eliminate the sex mismatching bias.

Fluorescence In Situ Hybridization

Dual-color fluorescence in situ hybridization (FISH) method was applied to the 5-µm thick archival formalin-fixed, paraffin-embedded tissue sections. Bacterial artificial chromosome (BAC) genomic clone RP11-349C2 was identified in the Resources for Molecular Cytogenetics database (www.biologia.uniba.it) by its location at the RECQL4 locus (chr8:145,707,623-145,713,976, UCSC genome browser, www.genome.ucsc.edu, version March 2006). The BAC clone was obtained from the Centre for Applied Genomics (Toronto, Ontario, Canada). The presence of the RECQL4 sequence and the correct chromosome location of the BAC clone were verified by PCR and by hybridization to metaphase spreads from normal peripheral lymphocytes, respectively. The BAC probe was labeled either with ULYSIS-Alexa-594 or with ULYSIS-Oregon-green-488 (Molecular Probes, Invitrogen) and was combined with the alpha satellite centromeric probe of chromosome 8, cen(8) (CEP 8 SpectrumGreen; Abbott Molecular, Des Plaines, IL) or with a commercial MYC-containing probe (LSI C-MYC-SpectrumOrange; Abbott Molecular), respectively. Dual-color FISH was performed according to standard procedures [38]. Either RECQL4 and cen(8) or RECQL4 and MYC were evaluated by spot visualization and enumeration for each probe in a range from 50 to 100 nonoverlapped, intact interphase nuclei per tumor tissue using a Zeiss Imager.Z1 microscope equipped with a digital camera AxioCam MRm and AxioVision 4.3 capturing software (Carl Zeiss Canada, Ltd., Toronto, Canada). If fluorescent signals could not be seen in at least 80% of cells, the result was considered to be noninterpretable (6/12). The most represented pattern of signal was recorded for the probe combinations mentioned above. The establishment of a cutoff value of >10% of tumor nuclei for the different probes used and for all signal patterns was defined considering the truncation artifacts, aneusomy, nuclear size, and chromatin condensation [39]. The relative gene copy number of RECQL4 was calculated by adding the number of RECQL4 signals to the RECQL4/cen(8) ratio. This calculation allowed to enhance the distinction between a ratio of 1 with two copies of each probe (2:2), and a ratio of 1 with three to five copies of each probe (3:3, 4:4, and 5:5). The pattern of signals (contiguous vs scattered) for the dual-color FISH experiment using RECQL4 and MYC probes was evaluated nucleus by nucleus to document the S-CIN of the 8q24 region. The observation of scattered signals was interpreted as the manifestation of S-CIN.

Chromosome copy number analysis was performed using centromeric enumeration probes for cen(3) (CEP 3 SpectrumRed), cen(7) (CEP 7 SpectrumGreen), and cen(17) (CEP 17 SpectrumAqua; Abbott Molecular). Sequential three-color FISH method was applied to the paraffin-embedded tissue sections according to the manufacturer's instructions. Chromosome enumeration was determined by scoring the number of signals for cen(3), cen(7), and cen(17) in 200 nonoverlapped intact interphase nuclei (10 of the 12 samples reached these criteria). The estimation of the overall ploidy of each sample is described case by case in the supplementary material (Table W2). Briefly, adapted from Rossi et al. [40], the OS cohort was classified by ploidy FISH as follows: 1) diploid, >50% of cells showing two signals for all probes; 2) near-triploid, >20% of cells showing three signals for at least two probes; 3) near-tetraploid, >20% of cells showing four signal for at least two probes; 4) polyploidy, >50% of cells showing more than five signals for at least one probe [40].

Results

RECQL4 Expression Levels in OS Determined by RT-PCR Analysis

To establish the expression level of RECQL4 in OS samples, we performed semiquantitative RT-PCR using total RNA extracted from patient tumors. We demonstrated that RECQL4 was overexpressed in all the tested tumors compared with normal osteoblasts. The mean expression levels observed for all 18 OS tumors was ∼13-fold higher than osteoblasts (range, x3 to x30; Figure 1). In keeping with the published literature, RECQL4 could not be detected in RNA-derived RTS fibroblasts [33,35]. OS1 exhibited the lowest level of RECQL4 expression, which was comparable to the low expression level observed in normal kidney tissue. In contrast, OS18 was characterized by an expression twice higher than that of the normal testis sample, which has been shown to be one of the most RECQL4-rich tissues [34]. We conclude that the expression level of RECQL4 is deregulated in OS.

Figure 1.

Figure 1

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RECQL4 is overexpressed in OS and is associated with S-CIN. The histogram is showing the fold change of RECQL4 mRNA compared with normal osteoblasts (NO) measured by semiquantitative RT-PCR. Rothmund-Thomson fibroblasts (RTS) and normal tissues (kidney and testis) were used as negative and positive controls, respectively. Each value is the result of three independent experiments. The SD is shown above each bar. The dashed line represents the mean value calculated from OS sample data. The lower panel part describes the chromosomal 8 instability (CIN) in the context of overall ploidy for 10 samples for which material was available for FISH studies. The absence of N-CIN or S-CIN is shown as (-). Identification of S-CIN or N-CIN is shown as (+). The overall ploidy status was established according to the FISH data using cen(3), cen(7), and cen(17). OS tumors that are showing an RECQL4 expression value close or more than the mean are showing S-CIN, whereas the ones less than the mean do not. The N-CIN and S-CIN are independent, and a high level of RECQL4 expression is associated with the appearance of S-CIN on the 8q24 region.

Association of RECQL4 Expression and Genome-wide Chromosomal Instability Levels

To determine whether there was a relationship between expression levels of RECQL4 and overall levels of CIN, we analyzed aCGH profiles to distinguish between N-CIN and S-CIN using 10 of the 18 tumors. Analysis of CNT distributions within each chromosome and the pattern of overall chromosomal imbalance present in each aCGH profile provided an objective overview of the variation in N-CIN and S-CIN levels that characterized each of the 10 OS analyzed (Table W1 and Figure 2). The mean number of N-CIN changes was 10.4 (SD, 5) for the study group, with no apparent relationship between the levels of RECQL4 expression and N-CIN (Figure 2A). In contrast, there was a clear trend showing an increase in S-CIN with higher expression levels of RECQL4. For example, OS18 had the highest expression level and exhibited >300 S-CIN aberrations and OS1 showed the lowest-expressing tumor for RECQL4 and had only 11 S-CIN aberrations. The mean number of S-CIN for the study group was 74.1 (SD, 95; Figure 2B). Collectively, these data indicate that elevated RECQL4 expression is associated with a greater incidence of CNTs and concomitant elevation in S-CIN frequency. We were not able to demonstrate any relationship between N-CIN levels and varying levels of gene expression of RECQL4.

Figure 2.

Figure 2

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Association between RECQL4 expression levels and varying N-CIN and S-CIN frequencies. (A) Differing frequencies of N-CIN (y-axis) as determined by analyzing imbalance profiles of whole chromosomes or entire arms for the 10 OS tumors (x-axis) after aCGH analysis. (B) Differing frequencies of S-CIN (y-axis) as determined by analyzing CNT distributions across each genomic profile derived from the ten OS tumors (x-axis) after aCGH analysis. The OS samples are ordered from left to right according to their RECQL4 expression level (lowest to highest) and are represented as a triangle in which the adjacent side represents the highest RECQL4 expression level.

Influence of RECQL4 Gene Copy Number on Its Expression in OS

The variation of the RECQL4 expression in OS could be the result of genomic copy number changes of the locus at 8q24. Dual-color interphase FISH analysis, using the RECQL4 and the cen(8) probes, was therefore performed on formalin-fixed, paraffin-embedded tissue derived from 12 OS tumors. As illustrated in Figure 3, A–D, and summarized in Table 2, we show that the increase of RECQL4 expression followed the genomic status of the RECQL4 gene. To determine copy number changes, cohybridization of RECQL4 and cen(8) probes and systematic analysis of the dual-color patterns allowed us to quantify numerical chromosome 8 abnormalities. OS samples with the lowest expression of RECQL4 (such as OS1 and OS4) have two normal chromosome 8s each bearing two copies of RECQL4 and cen(8) (Figure 3A). In OS2, four cen(8)s were present with only two copies of the RECQL4 gene, suggesting that a tetrasomy of chromosome 8 also involved RECQL4 loss (Figure 3B). OS5, OS7, and OS9 had a higher expression of RECQL4 (below the mean value) and three to five copies for each RECQL4 and cen(8) probes (Figure 3, C and D). This pattern could be interpreted as an acquisition of extra copies of an intact chromosome 8. Lastly, OS samples with the highest RECQL4 expression, (above the mean) all exhibited a copy number gain of RECQL4, which was observed as nine RECQL4 signals with four cen(8) for OS15 (Figure 3E) or six RECQL4 signals for two cen(8) as in OS13 (Figure 3F). Collectively, these findings indicate that of RECQL4 gene expression is largely copy number-dependent (Table 2).

Figure 3.

Figure 3

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(A–F) RECQL4/cen(8) dual-color interphase FISH experiment on OS tumors samples' fixed sections. RECQL4 probe is shown in red, whereas the cen(8) is in green. OS samples exhibited a normal pattern [OS1 (A)], a loss [OS2 (B)], an abnormal pattern associated with a 1:1 ratio [OS7 (C) and OS9 (D)], or a gain of RECQL4 [OS15 (E) and OS13 (F)]. (G–J) Structural CIN of the 8q24 cytoband. Dual-color interphase FISH was done on fixed tissue sections using the MYC probe (red) located on 8q24.21 and the RP11-349C2 probe (green) located on 8q24.3. The signal pattern of contiguous red and green signals on OS7 (G) and OS1 (H) identifies these samples as no S-CIN for the 8q24 cytoband. In contrast, for OS13 (I) and OS15 (J), the red and green signals are scattered all around the nuclei, characterizing these two samples as S-CIN for the 8q24 region.

Table 2.

RECQL4 Relative Gene Copy Number and Expression Analysis.

No. RECQL4 Copy Number (Genomic Status) cen(8) Copy Number RECQL4 Relative Copy Number* RECQL4 Expression (Fold Change)†
OS1 2 (N) 2 3 3
OS2 2 (L) 4 2.5 4
OS4 2 (N) 2 3 6
OS5 4 (AN) 4 5 6
OS7 3 (AN) 3 4 8
OS9 5 (AN) 5 6 10
OS11 6 (G) 4 7.5 14
OS12 3 (G) 2 4.5 14
OS13 6 (G) 2 9 15
OS15 8 (G) 4 10 22
OS16 9 (G) 2 13.5 23
OS18 5 (G) 3 6.6 30
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AN indicates aneusomy; G, gain; L, loss; N, normal.

*

RECQL4 relative number is calculated as described in the Materials and Methods section.

†

Expression of RECQL4 is normalized against the PBGD housekeeping gene expression, and shown as a fold change compared with the expression level measured in normal osteoblasts.

Influence of Structural Alterations of Cytoband 8q24 on RECQL4 Expression in OS

We used dual-color interphase FISH to study S-CIN levels within 8q24.21 (MYC probe) and 8q24.4 (RECQL4 probe) cytobands. Because both probes are closely linked within 8q24, paired two-color signals within nuclei can be used to determine whether disruption of the cytoband has taken place. In four OS tumors (OS1, OS4, OS5, and OS7), there was no evidence of disruption between MYC and the RECQL4 probes within nuclei (Table 3 and Figure 3, G and H). For six OS tumors, disruption of 8q24 was apparent. In OS13 (Figure 3I), six copies of 8q24 were apparent, but the red and green signals were no longer paired, indicative of structural rearrangement between the MYC and RECQL4 loci. In OS15 (Figure 3J), we observed the most complex signal pattern in the series; no pairing of green and red signals was apparent, and there was also evidence of numerical change. Thus, a range of structural aberrations affecting 8q24, varying from simple to complex, was apparent within this series of OS tumors. The varying levels of both N-CIN and S-CIN in the 10 OS as determined by FISH analyses of 8q24 with probes are detailed in Table 3. OS1 and OS4 did not exhibit numerical or 8q24 structural aberrations. OS5 and OS7 were both characterized by N-CIN only, whereas OS12 and OS13 had S-CIN only. The largest group of tumors, OS9, OS11, OS15, and OS18 had more complex FISH patterns, with both N- and S-CIN occurring together at 8q24. We then compared RECQL4 expression findings in tumors with N- or S-CIN at 8q24 to determine whether expression levels were associated with numerical or structural alterations of this region of chromosome 8. OS tumors with RECQL4 expression values that were close or higher than the mean value (13.5) across the 18 OS samples had S-CIN for the 8q24 region. This relationship held regardless of the N-CIN level or ploidy for chromosome 8 (Figure 1). Moreover, OS with the highest S-CIN levels determined by aCGH (shown above) also had the highest S-CIN level with the 8q24 cytoband region. These findings do not indicate that disruption of 8q24 leads to elevated expression of RECQL4 per se; rather, elevated RECQL4 is strongly associated with a greater overall frequency of S-CIN.

Table 3.

Relative Levels of Numerical and Structural Chromosomal Instability in 10 OSs.

No. Numerical Aberration Structural Aberration of Chromosome 8 Type of CIN for Chromosome 8
Overall Ploidy* cen(8) RECQL4 Copy Number MYC Copy Number Pattern of RECQL4 and MYC FISH Signals
OS1 Diploid 2 2 3 Not rearranged —
OS4 Diploid 2 2 2 Not rearranged —
OS5 Near-triploid 4 4 4 Not rearranged N-CIN
OS7 Near-triploid 3 3 3 Not rearranged N-CIN
OS9 Polyploid (>5n) 5 4 4 Scattered S- and N-CIN
OS11 Diploid 4 6 4 Scattered S- and N-CIN
OS12 Near-tetraploid 2 3 2 Scattered S-CIN
OS13 Diploid 2 6 6 Scattered S-CIN
OS15 Near-tetraploid 4 8 5 Scattered S- and N-CIN
OS18 Near-triploid 3 5 3 Scattered S- and N-CIN
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CIN indicates chromosomal instability; N-CIN, numerical chromosomal instability; S-CIN, structural chromosomal instability.

*

The overall ploidy has been estimated by the enumeration of cen(3), cen(7), and cen(17) (Table W2) as described in the Materials and Methods section.

Discussion

Previous studies of the RECQL4 gene have shown a strong association between constitutional mutations of the locus and predisposition to OS with 32% of RTS patient developing OS [22]. Moreover, fibroblasts and lymphocytes from RTS exhibit both N- and S-CIN [41–45]. Sequence analyses of RECQL4 in a large series of sporadic OS tumors failed to detect mutations in a significant proportion (only 3 samples of 71 had mutation/deletion) [46], but no study to date has determined the RECQL4 genomic status or analyzed expression levels in the context of CIN in OS.

The biologic function of the RECQL4 protein is poorly understood. The extensive studies of RTS, BLM and WRN, and other members of the RecQ helicase family, have not provided conclusive information concerning the specific and diverse functions of the RECQL4 protein. It is clear that for all three syndromes, germ line mutation of a specific RecQ helicase gene is associated with premature aging, CIN, and predisposition to diverse types of cancer [47]. However, there are important clinical and biologic differences between these genes. For example, conserved areas of the RECQL4 and BLM helicase motif do not have functional equivalence in vitro [27]. Each syndrome has distinct clinical features, and the associated cancer risk involves a different spectrum of tumors [48–50]. Moreover, the prominent class of CIN observed in the lymphocytes or fibroblasts of each syndrome varies, with RTS being characterized by chromatid breaks and isochromosomes, BLM by triradial and quadriradial figures, and WRN by multiple clones with distinctive balance translocations [50–52]. Interestingly, recent characterization of the overexpression of the RecQ helicases has shown it to be linked with the deregulation of the Rb pathway and the RAS activation. These data provide additional support to our supposition that there is an association between oncogenesis and RecQ helicases' overexpression [53]. In the proposed model, RecQ helicases' overexpression would promote and facilitate the DNA synthesis and telomere maintenance, processes that are mandatory for any transformed cells. However, similar to the different effects of the WRN, BLM, or RECQL4 mutations, the regulation of the RecQ helicase seems to be specific for each of the member [53]. In the last few years, several groups identified a more specific function for the RECQL4 helicase, specially its involvement in the early steps of replication fork machinery [27–30]. In an animal model, Sangrithi et al. [28] showed that the Xenopus laevi RECQL4 homolog is associated with chromatin during replication initiation and makes the origin of replication accessible to the replication factors. This function acts on the initiation and the unraveling of single-stranded DNA at the origin of replication. An altered expression of RECQL4 could disturb the processes governing the duration and extent of single-strand DNA exposure. Therefore, these single-stranded DNA regions would be the targets of DNA fragility and breaks. This abnormal single configuration of DNA is likely to promote interchromosome exchanges in a pseudo homolog recombination way. Because RECQL4 is activated by single-strand DNA and it promotes annealing of single-strand DNA to its complementary sequence, an excess of RECQL4 could force unmatched DNA annealing sequence, whereas a lack of RECQL4 could impair proper reannealing of separated strands of DNA [27]. Thus, the deregulation of RECQL4 could have profound consequences on overall genomic integrity and CIN in terms of structural complexity and heterogeneity.

By mapping the distribution of regions of imbalance and, in particular, determining the location of CNTs (Table W1) within each chromosome, it is possible to evaluate the relative contributions of N-CIN and S-CIN to destabilizing the OS genome. In this study, we have found that elevated RECQL4 expression is associated with a greater overall frequency of structural chromosomal change, but there was no obvious relationship between expression and N-CIN levels (Figure 2). Moreover, S-CIN changes were also apparent when FISH was used to determine the frequency of structural alteration at cytoband 8q24. It is possible that the elevated levels of RECQL4 would keep the DNA in a prolonged single stranded vulnerable stage, and promote the S-CIN. A result of this deregulation would be the perpetual initiation of S-CIN, leading to a polyclonal population of cells within one tumor. Indeed,OS tumor can exhibit highly polyclonal population where the most represented clone could account for only 30% of the cell [2–4].

Overexpression of RECQL4 has also been reported in other sarcomas (leiomyosarcoma, liposarcoma, and synovial sarcoma) and in some carcinomas (breast, colon, cervix, and laryngeal squamous cells), and its elevated expression has been correlated with metastasis or a later stage of disease [54–58]. Of these tumors, none have been studied systematically to determine whether S-CIN levels are elevated. However, in liposarcoma, the presence of a supernumerary giant chromosomes that varies structurally from cell to cell suggests ongoing instability [59]. These observations would give RECQL4 a strong impact in oncogenesis in general. In the present study, the MYC amplicon (8q24.21) seems to be independent of from the RECQL4 locus (8q24.3). The study by Mai and Mushinski [60] suggests that amplification and deregulation of MYC lead to instability characterized by gene amplification rather than by elevated frequencies of structural chromosomal rearrangement. This would be consistent with our finding: we observed a disruption of the 8q24 cytoband rather than an over-replication/amplification pattern (Figure 3, I and J), as one would expect as a result of an MYC-driven S-CIN in a given tumor. Furthermore, MYC is a multifunctional protein that acts on cell cycle, apoptosis, and cellular transformation through its transcription factor activity [20]. Conversely, RECQL4 seems to be more involved in specific DNA metabolism. We therefore suggest that the imbalances of the MYC oncogene and RECQL4 are two independent processes of oncogenesis.

In this study, we have investigated the relationship between S-CIN/N-CIN in OS and the RNA expression levels of RECQL4. We find that whereas ploidy changes and elevated N-CIN is common in OS, the more structurally abnormal tumors have higher levels of RECQL4. OS with the highest S-CIN levels determined by aCGH also had the highest S-CIN level with the 8q24 cytoband region. We found no evidence that disruption of 8q24 led to an elevated expression of RECQL4; rather, elevated RECQL4 is strongly associated with a greater overall frequency of S-CIN that characterizes the OS genome.

Supplementary Material

Supplementary Figures and Tables
neo1103_0260SD1.pdf (49.7KB, pdf)

Acknowledgments

The authors thank Olga Ludkovski (Ontario Cancer Institute, Toronto, Canada) for her technical assistance for the enumeration FISH experiments.

Footnotes

1

This research is supported by the Canadian Cancer Society 016215. G.M. is the recipient of the Helena Lam award.

2

This article refers to supplementary materials, which are designated by Tables W1 and W2 and are available online at www.neoplasia.com.

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