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Comprehensive and Definitive Molecular Cytogenetic Characterization of HeLa Cells by Spectral Karyotyping

Genome Technology Branch, National Human Genome Research Institute [M. M., E. S., H. P-N., B. M. G., T. R.], and Laboratory of Experimental Carcinogenesis, National Cancer Institute [C. K., D. Z., N. P.], NIH, Bethesda, Maryland 20892

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS SKY. CGH. HeLa Karyotype. FISH Localization of HPV18... HeLa Karyotype Comparison. DISCUSSION REFERENCES

 
We revisited the cytogenetic alterations of the cervical adenocarcinoma cell line HeLa through the use of spectral karyotyping (SKY), comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH). SKY analysis unequivocally characterized all abnormal chromosomes. Chromosomal breakpoints were primarily assigned by simultaneous assessment of SKY painted chromosomes and inverted 4,6-diamidino-2-phenylindole banding from the same cell. Twenty clonally abnormal chromosomes were found. Comparison with previously reported HeLa G-banding karyotypes revealed a remarkably stable cytogenetic constitution because 18 of 20 markers that were found were present before. The classification of 12 markers was refined in this study. Our assignment of the remaining six markers was consistent with those described in the literature.

The CGH map of chromosomal copy number gains and losses strikingly matched the SKY results and was, in a few instances, decisive for assigning breakpoints. The combined use of molecular cytogenetic methods SKY, CGH, and FISH with site-specific probes, in addition to inverted 4,6-diamidino-2-phenylindole or conventional G-banding analysis, provides the means to fully assess the genomic abnormalities in cancer cells.

Human papillomaviruses (HPVs) are frequently integrated into the cellular DNA in cervical cancers. We mapped by FISH five HPV18 integration sites: three on normal chromosomes 8 at 8q24 and two on derivative chromosomes, der(5)t(5;22;8)(q11;q11q13;q24) and der(22)t(8;22)(q24;q13), which have chromosome 8q24 material. An 8q24 copy number increase was detected by CGH. Dual-color FISH with a c-MYC probe mapping to 8q24 revealed colocalization with HPV18 at all integration sites, indicating that dispersion and amplification of the c-MYC gene sequences occurred after and was most likely triggered by the viral insertion at a single integration site. Numerical and structural chromosomal aberrations identified by SKY, genomic imbalances detected by CGH, as well as FISH localization of HPV18 integration at the c-MYC locus in HeLa cells are common and representative for advanced stage cervical cell carcinomas. The HeLa genome has been remarkably stable after years of continuous cultivation; therefore, the genetic alterations detected may have been present in the primary tumor and reflect events that are relevant to the development of cervical cancer.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS SKY. CGH. HeLa Karyotype. FISH Localization of HPV18... HeLa Karyotype Comparison. DISCUSSION REFERENCES

 
HeLa was derived from an adenocarcinoma of the cervix in 1952 and was the first human epithelial cancer cell line established in long-term culture (1) . The cells have a hypertriploid chromosome number (3n+), specific numerical deviations, 20 clonally abnormal chromosomes [known as HeLa signature chromosomes or HeLa markers (2) ], and contain multiple copies of the HPV⁴ type 18 (HPV18), integrated at specific sites (3) . These are stable characteristics following numerous years of weekly subculturing and provide a diagnostic tool for the detection of cross-contamination of cell cultures with HeLa cells.

HeLa cells have been used in a broad variety of investigations, including the identification of chromosomes and genes in somatic cell hybrids controlling the tumorigenicity in cervical carcinoma (4 , 5) . Because of the wide interest in this cell line, there has been a considerable effort over the years by experienced cytogeneticists to characterize in detail the chromosomes of HeLa and derivative lines. Thus far, G-banding and FISH with chromosome painting probes (2 , 4 , 6, 7, 8, 9, 10, 11, 12) have been the staining techniques of choice, but both methods have their limitations. Classical cytogenetic analysis of solid tumor cell lines often remained incomplete, because chromosome regions that appeared abnormal did not always have a characteristic band that could be accurately identified, and, in addition, the high number of aberrations impeded a characterization of all abnormal chromosomes. FISH with chromosome painting probes was mostly used as a confirmatory technique that required previous knowledge of cytogenetic abnormalities. Whole-genome analysis is time-consuming because it requires iterative experiments with different probes (12) . Now these methodological limitations can be overcome by 24-color FISH for the differential visualization of all 24 chromosomes in one experiment (13 , 14) .

Here, we applied advanced molecular cytogenetic techniques to the analysis of HeLa chromosomes and provided its complete and definitive cytogenetic profile. The data were generated by SKY (14 , 15) , concomitant inverted DAPI banding analysis, CGH for detecting changes in DNA copy number throughout the tumor genome (16) , and conventional FISH to verify the composition of complex marker chromosomes.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS SKY. CGH. HeLa Karyotype. FISH Localization of HPV18... HeLa Karyotype Comparison. DISCUSSION REFERENCES

 
HeLa Metaphase Preparation.
HeLa CCL2 cells were obtained at passage 90–102 from American Type Culture Collection (Rockville, MD) and have been stored in liquid nitrogen stocks. The same stocks were used before for G-banding karyotype analysis (6) and isotopic in situ hybridization for chromosome localization of HPV18 integration sites (3) . HeLa metaphase specimens were prepared according to standard cytogenetic procedures for solid tumors (17) .

SKY.
SKY is based on FISH, epifluorescence light microscopy, digital imaging, and Fourier transform spectroscopy (16) . The SKY hybridization protocol has been described in detail (14 , 18) . Essentially, metaphase cells of HeLa were pretreated and hybridized with a SKY probe mixture containing 24 uniquely labeled chromosome-specific probes. Probes were obtained by high-resolution flow sorting, amplified by two consecutive rounds of degenerate oligo-primed (DOP)-PCR amplification (19) , and were subsequently combinatorially labeled with three fluorochromes and two haptens to create a unique spectral signature for every chromosome. We used the fluorochromes Spectrum Green, Spectrum Orange (dUTP conjugates; Vysis, Downers Grove, IL) and Texas Red (12-dUTP conjugate; Molecular Probes, Eugene, OR) for direct labeling and biotin-16-dUTP and digoxigenin-11-dUTP (Boehringer Mannheim, Indianapolis, IN) for indirect labeling. After hybridization, biotin was detected with AvidinCy5 (Vector, Burlingame, VT), and digoxigenin-11-dUTP with mouse anti-digoxin (Sigma Chemical Co., St. Louis, MO), followed by sheep anti-mouse custom-conjugated to Cy5.5 (Amersham Life Sciences, Arlington Heights, IL). The slides were counterstained with DAPI and covered in para-phenylene-diamine antifade solution (Sigma).

Spectral images were captured with a SD200 SpectraCube system (Applied Spectral Imaging, Migdal Ha’Emek, Israel) mounted on a Leica DMRBE microscope (Leica, Wetzlar, Germany). Samples were illuminated with a 150 W Xenon lamp (Opti Quip, Highland Mills, NY) and imaged with a 63x/N.A.1.25 plan apo oil immersion objective (Leica) through a custom-designed triple bandpass optical filter (SKY v.3; Chroma Technology, Brattleboro, VT). Fourier transformation recovers the spectrum from this interferogram (16 , 20) . Spectrum-based classification of the raw spectral images is performed using SkyView 1.2 software (Applied Spectral Imaging). The visualization software applies an RGB look-up table to the raw spectral image resulting in the infra-red emission being displayed in red, red emission in green, and green emission in blue. The display image is a true representation of the SKY hybridization because it is based on the fluorescence intensities of all chromosome painting probes (Fig. 1A) . A mathematical classification algorithm is applied to unambiguously discriminate differentially labeled chromosomes based on their emission spectrum (16) and assigns to each pixel in the image with the same emission spectrum the same classification (pseudo-) color (Fig. 1, B and D) . DAPI banding gray-scale images of the same cells were captured separately, electronically inverted, and contrast-enhanced in SkyView 1.2 (Applied Spectral Imaging; Fig. 1C ). To define chromosomal breakpoints, the high-quality inverted DAPI banding images were aligned with the SKY classified chromosomes. Cytogenetic nomenclature adheres to ISCN (21) standards based on G-banding at the 400 band level.

View larger version (75K): [in this window] [in a new window]   Fig. 1. A, an RGB display of the 24-color SKY hybridization of a representative HeLa metaphase from subclone A (cell A1). The same metaphase in SKY classification colors (B), in gray-scale inverted DAPI banding (C), and arranged in a SKY karyotype (D) is shown. E, a previously published G-banding karyotype of HeLa from the same clone and passage as has been used for SKY analysis [figure used with permission, Popescu et al. (6)] and has been adjusted according to SKY results. Arrow, a possible deletion of 4q32qter as detected by CGH but not by SKY.

CGH.
CGH was performed on blood lymphocyte metaphases from a healthy female donor. Whole genomic DNA was extracted from blood lymphocytes and HeLa cells according to standard procedures (22) . CGH procedures have been described in detail previously (23) . In short, control and test genomes were differentially labeled by nick translation with, respectively, digoxigenin-11-dUTP and biotin-16-dUTP (Boehringer-Mannheim). Cohybridization with centromere-specific probes (24) for chromosomes 4, 8, 14, 20, 22, and X greatly facilitates chromosome classification critical for proper evaluation of CGH results. Hybridization of pretreated metaphase slides was performed for two nights at 37°C in a humidified chamber. Immunocytochemical detection of haptenized probes have been described previously (25) . Chromosomes were counterstained with DAPI and covered in para-phenylene-diamine (Sigma) antifade solution. For image acquisition, we used Leica QFISH software (Leica Imaging Systems, Cambridge, United Kingdom) interfaced to a cooled CCD camera (CH250; Photometrics, Tucson, AZ) and a Leica DMRXA microscope that was especially equipped for CGH image capture with four aligned optical filters for DAPI, FITC, TRITC, and Cy5 (TR1, TR2, TR3, and 41008; Chroma Technology). CGH ratio profiles were calculated on a Cytovision 3.0 workstation (Applied Imaging, Cambridge, UK) that was custom-upgraded for the display of the fourth image showing the Cy5 centromere ISH signals. The average ratio profile was calculated as a mean value of 15 metaphase spreads. Detection thresholds were 0.75 for copy number losses and 1.25 for copy number gains.

FISH.
To verify the composition of complex marker chromosomes, dual-color FISH was performed with painting probes for chromosomes 8 and 22 (M6, M16), chromosomes 7 and 19 (M8), and chromosomes 9 and 11 (M11), allegedly present in the markers. Chromosome preparations obtained from exponentially growing HeLa cells were hybridized with a HPV18 probe cloned from a cervical carcinoma biopsy used previously for isotopic in situ hybridization mapping of the virus in HeLa chromosomes (3) , genomic probes for c-MYC and c-ABL1 genes (Oncor, Gaithersburg, MD), and YAC 850A and a cosmid probe specific for region 3p14.2 [kindly provided by Dr. Kay Huebner (26 , 27) ]. FISH probes were labeled with biotin or digoxigenin by random priming (Boehringer-Mannheim) and visualized by FITC and TRITC, respectively. The conditions for FISH, digital image acquisition, processing, and analysis were performed as described previously (27 , 28) .

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS SKY. CGH. HeLa Karyotype. FISH Localization of HPV18... HeLa Karyotype Comparison. DISCUSSION REFERENCES

 
Molecular cytogenetic analysis of HeLa cells was performed by SKY, CGH, and FISH. SKY analysis always included inverted DAPI banding assessment of the same cell. The spectral images of a representative cell (A1) are shown in Fig. 1, A–D , and the SKY results from six fully analyzed HeLa cells are presented in Table 1 . A combined map of chromosomal gains and losses as detected with SKY and CGH is depicted in Fig. 2 . Both SKY and CGH contributed to the elucidation of the karyotype as demonstrated in Table 2 , classifying all chromosomal regions and breakpoints in rearranged chromosomes. The definitive HeLa karyotype is compared with karyotypes published previously (Table 3) to demonstrate the stability of this cell line over the years with respect to the presence of the numerous markers. Furthermore, FISH with gene-specific probes demonstrated colocalization of HPV18 with c-MYC on two marker chromosomes, M6 and M16 (Fig. 3A) .

View larger version (54K): [in this window] [in a new window]   Fig. 2. Chromosome idiograms with SKY amplification-deletion map (left of idiogram) and CGH ratio profile (right of idiogram). The SKY amplification-deletion map is based on 24 cells and is depicted as a color bar in which green indicates amplified regions, blue indicates modal (3n) regions, and red indicates deleted regions. Thick green bars, multiple amplified regions. Dotted bars, regions that were assigned with the help of CGH. Right, CGH average ratio profile; vertical lines, different values of the fluorescence ratio between tumor and normal reference DNA. The black middle line (r = 1.0) marks the balanced situation, the first red line on the left (r = 0.75) marks the threshold for copy number losses, and the first green line on the right (r = 1.25) marks the threshold for copy number gains. Note the remarkable consistency between the SKY amplification-deletion map and the map for CGH gains and losses.

View larger version (54K): [in this window] [in a new window]   Fig. 3. A, the HPV18 (FITC, green) and c-MYC (TRITC, red) fluorescent signals that colocalized (yellow) on HeLa metaphase chromosomes (DAPI, blue) on normal chromosomes 8 at 8q24 and on two abnormal chromosomes, M6 and M16, now known to contain chromosome band 8q24 as well. B, HeLa metaphase chromosomes (propidium iodide, red) with the FHIT/FRA3B locus-specific FISH signals (FITC, yellow) mapping to 3p14.2 on one normal chromosome 3 and two abnormal chromosomes, M4 and M10.

	SKY.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS SKY. CGH. HeLa Karyotype. FISH Localization of HPV18... HeLa Karyotype Comparison. DISCUSSION REFERENCES

Gray-scale images of the DAPI counterstain from the same cells were electronically inverted (Fig. 1C) and aligned with the spectrally analyzed chromosomes to define the chromosomal breakpoints. Common chromosomal breakpoints in subclones A and B were localized at 9 centromeric regions, 1p10, 3p10, 3q10, 5p10, 5q10, 9p10, 13q10, 15q10, and 20q10, and at 25 chromosome bands, 1q11, 2q31*, 2q36*, 3p11, 3q11, 3q21, 5q11, 7p21, 7q35, 8q24*, 9p11, 9q34*, 11p11*, 11p14*, 11q13*, 11q22*, 12q15, 13q12, 13q14, 13q21, 16p11, 19p13*, 19q11, 22q11, and 22q13. In some cases, we were not able to determine the exact chromosomal breakpoints by SKY and inverted DAPI banding alone. However, in cases where a recurrent aberration resulted in a genetic copy number change, the CGH ratio profile was indicative for the breakpoints. The breakpoints marked with an asterisk denote those that were identified using CGH (Table 2) . Several of these regions are involved in recurrent aberrations associated with cervical cancer and other malignancies.

	CGH.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS SKY. CGH. HeLa Karyotype. FISH Localization of HPV18... HeLa Karyotype Comparison. DISCUSSION REFERENCES

 
CGH is a single-experiment screening test used to map gains and losses in a tumor genome and to localize potential sites of proto-oncogenes and tumor suppressor genes. The average ratio profile in Fig. 2 shows the chromosomal regions in HeLa with a decreased or increased copy number (see the figure legend for details). We found copy number gains of chromosome 1, chromosome arms 5p, 9p, 15q, 16p, and 20q, and regions 2q31q36, 3pterp21, 5q11q32, 7pterq35, 8q24qter, 9q34qter, 11p14p11, 11q13q22, and 12pterq15. Copy number losses were detected for chromosome arm 20p and regions 4q32qter, 7q35qter, 11q23qter, and 13q10q14 (Table 2) .

	HeLa Karyotype.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS SKY. CGH. HeLa Karyotype. FISH Localization of HPV18... HeLa Karyotype Comparison. DISCUSSION REFERENCES

 
The results of SKY, CGH, and FISH analyses of HeLa cells are combined and described in detail in the following section. CGH was primarily used to complement SKY. From SKY and previous cytogenetic studies, HeLa was found to be a hypertriploid cell line (3n+). Therefore, CGH detected copy number gains for chromosome regions if they were present in more than three copies in the majority of cells and mapped copy number losses for regions if they occurred in fewer than three copies. Fig. 2 depicts the combined SKY and CGH results aligned next to their respective chromosome idiograms. SKY amplification-deletion is depicted as a color bar on the left side of the chromosome ideograms, and CGH gains and losses are shown as average ratio profiles on the right side of each chromosome idiogram (see the figure legend for details). Table 2 summarizes the number of normal chromosomes present, the regions present in the markers, and the total sum of chromosomal material for chromosomes 1–22 and the X as assessed by SKY and inverted DAPI banding analysis. Furthermore, Table 2 lists the gains and losses as detected by CGH and additional FISH data verifying and refining the SKY results. The definitive karyotype resulting from the combined SKY, inverted DAPI banding, and CGH and FISH analyses is given in Table 3 .

HeLa contained three normal chromosomes 1 and two clonal markers, M1 der(1)t(1;3)(q11;q11.2) and M2 der(1;9)(p10;q10), resulting in tetraploidy for chromosome 1. In Fig. 2 , the green bar left of the idiogram represents the extra chromosome 1 material in M1 and M2 as detected by SKY. This was in concordance with the CGH average ratio profile for chromosome 1 showing a gain for both the p and q arms.

There were two normal chromosomes 2 present and one clonal marker (M3) that had an elongated 2q arm. SKY and inverted DAPI banding could not determine which region of chromosome 2 was amplified because of its undefined chromosomal banding pattern. CGH, however, suggested a duplication of 2q31q36. In Fig. 2 , this region is indicated by a green dotted bar.

Chromosome 3 had one normal copy and was involved in six clonal markers (M1, M4, M5, M10, M12, and M20). M1 and M5 were whole-arm translocations both containing 3q, producing a triploid copy number for 3q. M4 and M10 both contained the whole p arm, adding up to a triploid copy number for 3p as well. M12 possibly contained 3q21qter, resulting in one copy gain for this region. CGH showed a balance for 3p14qter and gain of 3p21pter, suggesting that 3p21pter was present in marker 20. FISH with a cosmid probe spanning the FRA3B/FHIT locus at 3p14.2 demonstrated hybridization signals for the probe on the normal copy of 3, M4, and M10 but not on M20 (Fig. 3B) .

Chromosome 4 usually had three normal copies. CGH suggested a loss of 4q32qter, whereas SKY and inverted DAPI banding did not show the deletion. In the Giemsa-stained karyotype (Fig. 1E) , there was one chromosome 4 (arrow) that appeared shorter and may have a small deletion. Additional high-resolution G-banding or FISH analysis with 4q telomere-specific probes would perhaps resolve this discrepancy.

Two normal chromosomes 5 were present in HeLa in addition to three clonal markers (M4, M6, and M7). M4 and M6 each contained the whole 5q arm, including the centromere, resulting in a gain of 5q material. M7 was an isochromosome 5p and was present in three to five copies. Furthermore, some cells had nonclonal markers that contained 5p (M22 and M27). We have detected up to 12 copies of 5p in one cell. CGH revealed a gain of 5q and a high copy number gain of 5p.

HeLa usually contained two normal chromosomes 7, two clonal markers (M8 and M9), and two markers present in subclones A and B that are mutually exclusive (M19 and M20). M8 is a der(7)t(7;19) with 7pter7q35 present. M9 contained part of 7p, possibly 7pterp21, translocated to 16p. M19 and M20 are derivatives 7, both containing 7p21qter. Chromosome 7 material in these markers was confirmed by conventional FISH. In total, the SKY amplification-deletion map showed a gain for 7pter7q35 and a balance for 7q35qter. This was partly reflected in CGH, which showed a gain of 7pter7q35 and a loss for 7q35qter. The loss of 7q35qter was perhaps due to the presence of subpopulations of HeLa containing fewer than two normal chromosomes 7.

Chromosome 8 was represented by three normal copies and two clonal markers (M6 and M16). In a previous study, the markers M6 and M16 were found to contain HPV18 sequences, but their origin could not be elucidated by G-banding (3) . SKY revealed a piece of chromosome 8 at the telomeric ends, which was confirmed by conventional FISH (data not shown), although the exact banding region could not be determined by this method. However, CGH indicated a gain of 8q24qter, the site of the c-MYC proto-oncogene, suggesting there were extra copies of the c-MYC gene. Remarkably, dual-color FISH with HPV18 and c-MYC-specific probes revealed 100% colocalization on normal chromosome 8 and on M6 and M16, verifying that 8q24 was present in both markers (Fig. 3A) .

Chromosome 9 was usually represented by two normals and three clonal markers (M2, M10, and M11). M2 contained 9q, and M10 contained 9p, including the centromere, hereby balancing chromosome 9 material. Conventional FISH with chromosome 9 and c-ABL1 probes has confirmed the presence of chromosome 9 material at both distal ends of M11 and detected the presence of five copies of the c-ABL1 gene (data not shown). CGH showed a distinct gain of 9q34qter. SKY identified two nonclonal markers containing the 9p arm (M22 and M23). It is conceivable that HeLa is heterogeneous with regards to 9p markers because CGH showed a gain for 9p.

HeLa had two normal copies of chromosome 11 and one clonal marker (M11) containing a complex intrachromosomal rearrangement that remained elusive after inverted DAPI and G-banding analysis. CGH indicated amplification of 11p14p11 and 11q13q22 and loss of 11q23qter.

The average number of normal chromosomes 12 was three. One clonal marker (M12) contained 12pterq15, thereby resulting in an overall gain for this region. Accordingly, CGH showed an increased copy number for 12pterq15 and was balanced for 12q15qter.

Chromosome 13 was present as two normals copies in subclone A and ranged from zero to two normals in subclone B. Marker M14 contained 13q21qter and was present in both subclones. M17, an isochromosome 13q with deletions in both arms at 13q12 and 13q14, respectively, is subclone B specific. On the basis of the SKY analysis of subclone A alone, one would expect a loss for 13q10q21 and a balance for 13q21qter, as shown by CGH. The contribution of chromosome 13 material from subclone B-specific marker M17 did not balance the region 13q10q14, because subclone B often showed concomitant loss of normal chromosomes 13.

Chromosome 15 was present in two normal copies, one clonal marker isochromosome 15q (M13), and one subclone B-specific marker also containing 15q (M18), contributing to four or five copies/cell. CGH showed a gain of 15q.

HeLa cells always contained three normal chromosomes 16 and one clonal marker (M9) containing 16p. As expected, CGH showed a balance for 16q and a gain for 16p.

Chromosome 17 was commonly present as four normal copies; therefore, CGH should display a copy number gain. However, only a slight increase in copy number gain was detected, suggesting that tetraploidy for this chromosome was not present in >50% of the cell population. Previous G-banding studies have usually identified three chromosomes 17.

Two or three normal copies of chromosomes 18 were present in HeLa. CGH showed a slight loss of 18, reflecting the loss of 18 in a subpopulation of cells.

Chromosome 19 was represented by two normals and two clonal markers (M8 and M14). In M8, a small piece of 19 was present, as confirmed with conventional FISH, but its band origin remained elusive. Because M14 contained 19p13qter and CGH is balanced for 19, the unknown piece of M8 possibly originated from 19p13pter.

Chromosome 20 was present as two normals and two clonal markers (M5 and M15). M5 contained chromosome arm 20q, and M15 was an isochromosome 20q, resulting in a total of five copies of 20q and two for 20p. CGH showed a gain of 20q and a loss of 20p.

Subclone A usually contained three normal chromosomes 21 and no clonal markers. Subclone B contained two normals and one subclone B-specific marker (M18), which was a Robertsonian translocation, der(15;21). The total copy number of chromosome 21 equaled 3, and CGH showed a balanced ratio profile.

Chromosome 22 was present as two normal copies and two clonal markers (M6 and M16). M6 was a complex unbalanced translocation involving chromosomes 5, 8, and 22 and contained 22q11q13, and M16 was a der(22)t(8; 22) with the breakpoint in 22q13 as well. Conventional FISH confirmed the presence of chromosome 22 material in both markers (data not shown). On the basis of SKY, a copy number gain was expected for 22q11q13 and a copy number loss for 22q13qter. However, CGH showed a balance for this region, which might be due to the limited spatial resolution of CGH.

Chromosomes 6, 10, 14, and X each had three normal copies and were not involved in clonal markers. Accordingly, the CGH ratio value was r = 1.

	FISH Localization of HPV18 Integration Sites.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS SKY. CGH. HeLa Karyotype. FISH Localization of HPV18... HeLa Karyotype Comparison. DISCUSSION REFERENCES

 
We detected HPV18 sequences by FISH on chromosome 8 and two abnormal chromosomes identified previously as derivatives 5 and 22. These results were consistent with radioactive ISH analyses (3) . The derivation of these two aberrant chromosomes has been resolved by SKY (Table 1) . Both markers contained material from chromosome 8, and CGH displayed a gain of region 8q24qter. HeLa chromosomes were cohybridized with HPV18 and c-MYC probes to determine the copy number of these genes. Fifty complete metaphases were analyzed and in all of the spreads; HPV18 and c-MYC signals were detected at five sites as closely spaced or overlapping signals (Fig. 3A) . On the basis of the size of the fluorescence hybridization signals, we estimated 5–10 copies of HPV18 per integration site. The HPV18 and c-MYC hybridization signals were inseparable in interphase nuclei with diffuse and relatively decondensed chromatin as well as in prophase and prometaphase nuclei. Colocalization was detected in 8q24 on three normal chromosomes 8 and at the distal end of the p arm of M6 and the distal end of the q arm of M16, where the chromosome 8 material was located in both markers. The presence of c-MYC in M6 and M16 confirmed the presence of 8q24.

	HeLa Karyotype Comparison.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS SKY. CGH. HeLa Karyotype. FISH Localization of HPV18... HeLa Karyotype Comparison. DISCUSSION REFERENCES

 
We have compared the HeLa spectral karyotype with karyotypes published previously that were analyzed by G-banding (2 , 4 , 6, 7, 8, 9, 10, 11) and FISH (12) . Table 3 shows 31 HeLa markers characterized in this study. In our comparison, we chose to avoid karyotypes of different sublines and variations of HeLa, HeLa-like cell lines, and HeLa-contaminated cell lines. Nevertheless, a comparison with HeLa S3 and HeLa-variation D98/AH-2 was made for historical perspective.

The genome of HeLa has remained stable after decades of weekly subculturing. We found that 18 of 20 clonal markers (M1–M17 and M19) described by SKY were present in G-banding studies published previously. Two markers (M18 and M20) were not noticed or were not present before. Six of the 18 markers (M1, M2, M4, M7, M13, and M14) were described in the same way in at least one previous study, whereas 12 markers (M3, M5, M6, M8, M9, M10, M11, M12, M15, M16, M19, and M20) were identified in part or not at all. SKY and additional molecular cytogenetic techniques have fully identified these markers. One of the 12, M8 der(7)t(7;19)(q35;p13), was never recognized as being aberrant in the G-banding karyotypes. M8 was possibly present in three studies (6 , 8 , 9) but classified as a normal seven instead. M6 der(5)t(5;8;22) was recognized as being aberrant only by Popescu et al. (3 , 6) and Chen (2) ; in all other reports, it was classified as a normal chromosome 5. Markers M22 and M23 were present in previous G-banding (2 , 6) studies and may, therefore, be of clonal origin. M22 was identified in D98/AH-2 by FISH (12) . Nelson-Rees et al. (10) have given the description of markers found in HeLa CCL2 and HeLa S3 but did not show a full karyotype with the normal chromosomes. Therefore, there might be more common markers present than the six (CCL2) and five (S3) listed in Table 3 . We found that HeLa CCL2 had six markers in common with D98/AH-2, which was studied by FISH with chromosome painting libraries (12) and G-banding (4 , 8 , 11) . The discrepancies between our study and FISH whole-chromosome painting by Ruess et al. (12) merely exist because D98/AH-2 is a subline that is cytogenetically different from HeLa CCL2.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS SKY. CGH. HeLa Karyotype. FISH Localization of HPV18... HeLa Karyotype Comparison. DISCUSSION REFERENCES

 
The definitive karyotype of HeLa was resolved by combining the molecular cytogenetic techniques SKY, CGH, and FISH. The origins of all complex rearrangements previously unidentified in G-banding studies have been resolved. Comparison with previous G-banding studies revealed HeLa to be remarkably stable after years of cultivation because 18 of 20 clonal markers found in HeLa were present in earlier studies (Table 3) .

SKY and CGH analyses together have unprecedented power for resolving structural and numerical chromosomal aberrations. Twenty-four-color FISH analysis by SKY readily identifies the origins of rearranged chromosomes. When uncertainty remained after SKY about the origin of subtle translocated regions e.g., at a telomeric end or regions spanning less than one chromosome band, conventional FISH was used to establish the origin by using a flow-sorted whole chromosome painting probe. In this study, all confirmation experiments verified the initial identification of chromosomal material based on SKY. Alignment of the SKY-classified chromosomes with the companion inverted DAPI banding image was used to identify the chromosome band regions. Although the inverted gray-scale images of high quality DAPI-stained chromosomes were similar to G-banding, the banding resolution of HeLa metaphases in this study were sometimes suboptimal, making accurate breakpoint mapping difficult. CGH maps overall chromosomal copy number changes (gains and losses) of the tumor DNA over the length of the chromosome. In principal, therefore, any clonal aberration that causes a numerical imbalance is detected by CGH. In the few instances where SKY and inverted DAPI banding analysis were unsuccessful in identifying the origin of the chromosomal material involved, the CGH copy number change was indicative. A clear example of karyotype refinement by CGH was demonstrated for M3 dup(2q), where the extra material was determined to originate from the gained region in CGH, 2q31q36. There is striking similarity between the SKY amplification-deletion map and the CGH ratio profile, considering that the SKY analysis was based on 24 metaphase cells, whereas CGH analysis was based on DNA extracted from millions of cells. The clonal aberrations as described by SKY in the two subclones are representative for the entire HeLa population. This view was strengthened because 18 of 20 markers identified by SKY were present in the G-banding studies reported more than 10 years ago (Table 3) .

Balanced translocations, as detected frequently in hematological malignancies, were not identified in HeLa cells. Gain of function of proto-oncogenes and loss of function of tumor suppressor genes is mostly achieved via copy number changes that are detectable cytogenetically. Of course, activation or inactivation by point mutations remains elusive. The gain of chromosome arm 5p was mapped frequently in advanced stage cervical squamous cell carcinomas (29) and in other cell lines established from cervical cancers.⁵ SKY analysis of HeLa chromosomes identified the formation of three to five isochromosomes 5p as one of the underlying cytogenetic mechanisms by which chromosome arm copy number increases are acquired. The amplification of the entire short arm of chromosome 5 suggests the presence of genes whose gain of function seems to be highly advantageous in advanced stage cervical carcinomas and in cervical carcinoma cell lines.

In a relatively limited number of cases of cervical carcinoma cell lines or tumor samples examined, HPV was found integrated at apparently normal chromosomal sites (30) . In contrast, current FISH analysis demonstrated colocalization of HPV18 and c-MYC sequences in two rearranged sites. This strongly indicates genomic reorganization caused by the viral insertion and is consistent with a statistical analysis showing a correlation between the breakpoints in structural rearrangements and HPV sites of integration in cervical carcinomas (31) .

The complex marker M11 was present in all previous G-banding studies (Table 3) but has never been fully described before. SKY and additional FISH using chromosome painting probes for chromosomes 9 and 11 and c-ABL1-specific probes revealed M11 to contain chromosome 11 material and 9q34 segments on both distal ends. The HPV18 integration at 9q34 (3) was not confirmed in this study. The exact rearrangement of chromosome 11 segments in M11 could not be derived from SKY, FISH, or conventional cytogenetic techniques in this study. CGH analysis suggested, however, a deletion of 11q23qter, possible deletion of 11p15pter, and amplification of 11p14p11 and 11q1311q22. HeLa furthermore contained two normal chromosomes 11. On the basis of chromosome microcell transfer studies of chromosome 11 into HeLa cells (32) , it has been suggested that chromosome 11 contains tumor suppressor genes. LOH studies on cervical cancer localized a putative site to 11q22q24 (33) . This is in accordance with our CGH findings showing loss of 11q23qter. Additional FISH for chromosome band 11q13, containing the cyclin D1 gene, is necessary to elucidate presence of this region in HeLa because this region has been found to be involved in multiple translocations in HeLa sublines such as DH98/AH-2 (34 , 35) .

In conclusion, we reported here the most complete chromosomal characterization of the HeLa cervical cancer cell line. Numerical and structural chromosomal aberrations identified by SKY and inverted DAPI banding analysis, genomic imbalances detected by CGH as well as FISH localization of HPV18 integration at a fragile site and c-MYC locus in HeLa cells are common and representative for advanced stage cervix squamous carcinomas (29 , 36 , 37) . Thus, genomic alterations in HeLa cells are highly relevant to the pathology of cervical neoplasia and provide a reference for future studies in cervical cancer.

	ACKNOWLEDGMENTS

 
We thank Dr. S. du Manoir and Dr. K. Heselmeyer-Haddad (Genome Technology Branch, National Human Genome Research Institute, NIH, Bethesda, MD) for assistance with CGH analysis and helpful discussions; M. van der Linden and D. Laferty (Leica Imaging, Cambridge, United Kingdom) for adapting QFISH image acquisition to CGH; and M. Gregson (Applied Imaging, Cambridge, United Kingdom) for implementing Cy5 display into Cytovision 3.0 CGH analysis software. Image acquisition and analyses software was developed under NIH-CRADA terms with Applied Spectral Imaging, Ltd., Applied Imaging, Ltd., and Leica Imaging Systems, Ltd.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Laboratory for In Situ Hybridization, Institute of Pathology, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, the Netherlands. E-mail: m.macville{at}pathol.azn.nl

² Received a fellowship from the Deutsche Krebshilfe.

³ To whom requests for reprints should be addressed, at Genome Technology Branch, National Human Genome Research Institute, NIH Building 49, Room 4A28, 49 Convent Drive, Bethesda, MD 20892-4479. Phone: (301) 594-3118; Fax: (301) 435-4428; E-mail: tried{at}nhgri.nih.gov

⁴ The abbreviations used are: HPV, human papillomavirus; FISH, fluorescence in situ hybridization; SKY, spectral karyotyping; DAPI, 4,6-diamidino-2-phenylindole; CGH, comparative genomic hybridization; TRITC, tetramethylrhodamine isothiocyanate.

⁵ M. Macville et al., manuscript in preparation.

Received 6/25/98. Accepted 10/29/98.

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Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS SKY. CGH. HeLa Karyotype. FISH Localization of HPV18... HeLa Karyotype Comparison. DISCUSSION REFERENCES

 

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