The Hematology Journal (2004) 5, 61–68 & 2004 The European Hematology Association All rights reserved 1466-4680/04 $25.00 www.nature.com/thj
Use of polymorphisms in the noncoding region of the human mitochondrial genome to identify potential contamination of human leukemia-lymphoma cell lines Grit Hutter*1, Christina Nickenig1, Henk Garritsen2, Frank Hellenkamp3, Andre Hoerning3, Wolfgang Hiddemann1 and Martin Dreyling1 1
Department of Medicine III, University Hospital Grosshadern/LMU, GSF-Clinical Cooperative Group Leukemia, Germany; Director blood Transfusion services, Sta¨dtisches Klinikum Braunschweig, Germany; 3Department of Transfusion Medicine and Transplantation Immunology, University Hospital Muenster, Germany 2
The availability of the complete sequence of human mitochondrial DNA (mtDNA) has proven extremely useful in phylogenetic studies, forensic science and the determination of chimerism after allogeneic stem cell transplantation. In this study, we could demonstrate that the analysis of mtDNA polymorphisms is a quick and reliable method to identify contamination of human hematopoietic cell lines. This assay is based on PCR-sequencing of three hypervariable segments of the control region of mtDNA (hypervariable region (HRV) 1, 2 and 3). All three regions contain a large number of single-base polymorphisms. mtDNA was isolated according to standard laboratory procedures and amplified by PCR. Subsequently products were sequenced and evaluated with a semiautomated DNA sequencer system. So far, 21 human leukemialymphoma (LL) cell lines and nine other human cell lines were screened for contamination by other cell lines applying this method. We conclude that analysis of mtDNA polymorphisms is a quick, reliable and inexpensive method to detect intra - and interspecies cross-contamination and for the authentication of human LL cell lines. In comparison to other methods (cytogenetics, fluorescence in situ hybridization or immunophenotyping), this technique is less laborious and time consuming. The Hematology Journal (2004) 5, 61–68. doi:10.1038/sj.thj.6200317 Keywords:
mtDNA; hypervariable region; D-loop; cell line authentication
Introduction Human leukemia-lymphoma (LL) cell lines represent an important research tool to analyze the molecular pathogenesis of hematopoietic malignancies.1 However, analysis of LL cell lines may be hampered by frequent contaminations of microorganisms (particularly mycoplasmas in 20–30%) or other cell lines.2–5 Although it has been previously discussed,3–5 the latter problem does not yet appear to be sufficiently recognized. It is estimated that more than one-third of cell cultures in use are cross-contaminated either by inter- or intraspecies contamination.4,6 Methods used for cell line authentication are (1)
the traditional method of isoenzyme analysis, which detects interspecies contamination of at least 10%;7
*Correspondence: G Hutter, GSF-Haematologikum, KKG-Leukemie, Marchioninistr. 25, 81377 Munich, Germany; E-mail:
[email protected] Received 3 March 2003; accepted 2 June 2003
(2) (3)
DNA fingerprinting based on the specific pattern of polymorphisms of individual DNA samples (exclusion rates of 99% or higher);8 cytogenetic analysis (karyotyping), which is currently considered the most sensitive method for the identification of intraspecies contamination. However, it is a labor-intensive, time-consuming and a rather expensive procedure.8
Mutations analysis of mitochondrial DNA (mtDNA) has been used in population and evolutionary genetics as well as forensic identifications, medical diagnoses and mutation studies. Mitochondria are eucaryotic cytoplasmic organelles, which carry out oxidative phosphorylation, the main energy source of the cell. The circular 16.6 kb completely sequenced human mtDNA encodes 13 protein subunits of the oxidative phosphorylation complexes, the 12S and 16S ribosomal RNA and the 22 tRNAs required for mitochondrial protein synthesis.9,10 mtDNA contains also a noncoding region, called Dloop, that consists of regulatory elements for replication
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and transcription.11 This D-loop region is highly variable12,13 with a high mutation rate compared to genomic (nuclear) DNA.14 Garritsen et al.15 focusing on two highly variable regions (HRV1, HRV2) in the Dloop of the mtDNA could demonstrate that specific polymorphisms in this hypervariable regions can be used to distinguish individuals even in a relatively homogenous population of Caucasian blood donors. We therefore postulated that specific polymorphisms in the regions HRV1, HRV2 and HRV316 could be used for the authentication of human cell lines. In the present study, we applied mitochondrial PCR analysis on 30 different human cell lines, including 21 LL cell lines and nine other human cell lines. We amplified the three hypervariable segments of the control region of mtDNA: hypervariable region 1 (HRV1), 2 (HRV2) and 3 (HRV3) using previously published primers.15 Sequence analysis of these three regions revealed polymorphisms specific for each cell line. We could also demonstrate that PCR with humanspecific primers for HRV1, HRV2 and HRV3 and primers specific for the mouse mtDNA D-loop region reliably discriminated between human and mouse cell lines.
Materials and methods mtDNA isolation mtDNA from cell lines was isolated from 106 to 107 cells using the NucleospinR Blood XL kit (Machery-Nagel, Dueren, Germany).
Amplification and sequencing of mtDNA The primer sequences used for PCR amplification and sequencing are listed in Table 1. The PCR reaction for all five primer sets was performed in a 25 ml volume containing 10 pmol of each primer, mtDNA (1 ng), 0.25 mM of each dNTP, 1.25 U of Taq polymerase and 2.5 ml 10 reaction buffer (Amplitaq Gold, Perkin Elmer, Weiterstadt, Germany), 2.5 mM MgCl2, 5% dimethyl sulfoxide (DMSO; Merck, Darmstadt, Germany). PCR amplification was performed under identical amplification conditions for all primer sets (10 min initial denaturation and enzyme activation at 951C, followed by 30 cycles of denaturation at 941C for 30 s, annealing at 561C for 60 s and extension at 721C for 60 s). Amplified products (5 ml) were separated on a 2% agarose gel and visualized under ultraviolet illumination after ethidium bromide staining.
Automated sequencing A 1–2 ml aliquot (1 : 10 diluted in H2O) was sequenced using 2–4 ml of the ABI Big Dye Terminator cycle sequencing kit (Perkin Elmer, Weiterstadt, Germany) with 5 pmol of each sequencing primer in a total volume The Hematology Journal
of 10 ml. Each amplification was performed with the following thermal profile: 25 cycles of denaturation at 961C for 10 s, annealing at 501C for 5 s and elongation at 601C for 4 min. The purified products were sequenced on the ABI310 sequencer with the standard Amplitaq FS dye terminator protocol. Forward and reverse primer sequencing was performed to identify all bases. Data analysis: mtDNA analysis was carried out using the standard nucleotide–nucleotide blast and BLAST 2 sequences package (http://www.ncbi.nlm.nih.gov/BLAST/). Sequences were aligned to the human reference sequence 4gi|13959823|ref|NC_001807.3| Human mitochondrion, complete genome and the mouse reference sequence 4gi|342520|gb|J01420.1|MUSMTCG Mouse mitochondrion, complete genome. If nucleotides at a certain position differed from these reference sequences, they were considered as polymorphisms.
Results PCR amplification of mtDNA mtDNA was isolated from 30 human cell lines and three mouse cell lines. Figure 1 shows a representative amplification of mtDNA. All of the amplicons were of the expected size. In addition, specific primers of the HRV regions of human and mouse mtDNA (Table 1) discriminated between DNA derived from human or mouse cell lines (Figures 1 and 2). In human cell lines (6 106 cells/ml) a contamination with mouse cells down to 1.32 102 cells/ml could be reliably detected using specific primers of the HRV mtDNA region (Figure 3). The nature of PCR fragments was confirmed by sequencing.
Sequencing results of HRV1 DNA sequencing of the HRV1 region on 30 cell lines revealed 29 distinct genotype sequences. Two cell lines (PatuT and Patu S) were cell lines derived from the same individual, which are not expected to differ in their HRV1 sequence (Table 2). Each of the other cell lines showed a unique HRV1 sequence. These data strongly suggest that human cell lines can be accurately discriminated by their highly polymorphic HRV1 sequence. In comparison to the reference sequence (4gi|13959823|ref|NC_001807.3| Human mitochondrion, complete genome), no polymorphism was detectable in the HRV1 region in the cell line U937. In contrast in other cell lines, up to eight polymorphisms (Mec-1) were found (Table 2). In most cell lines, three (11/30) or four (8/30) characteristic polymorphisms were identified (Table 2). The most frequently detected polymorphisms were pyrimidine transitions at position 16069 C-T (5/30), 16126 T-C (5/30), 16223 C-T (5/30), 16224 C-T (5/30), 16311 T-C (4/30) and 16362 T-C (5/30).
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Figure 1 PCR amplification products of human (lanes 1, 3–5, 7–9, 11 and 12) and mouse cell line (lanes 2, 6 and 10) with the humanspecific mtreg 1 primers (lanes 9–12; expected size 521 bp), mtreg2 primers (lanes 5–8; expected size 422 bp) and mtreg3 primers (lanes 1– 4; expected size 541 bp). While mtreg3 primers were highly specific for human cell line DNA (no amplification with mouse template DNA, lane 2) the primer sets mtreg1 and mtreg2 displayed a weak amplification product from mouse template DNA (lanes 6 and 10). All PCR products from human template DNA were verified by cycle sequencing. Lane 13 – DNA 100 bp ladder.
Figure 2 PCR amplification products of mouse (32D lanes 2, 6; Baf3 lanes 4, 8) and human cell lines (Raji lanes 1, 5; JVM2 lanes 3, 7) with the mouse-specific mtmouse1 (lanes 1–4) and 2 (lanes 5–8) primers. Lanes 2, 4, 6 and 8 display the amplified product of mouse cell lines, whereas (lanes 1, 3, 5 and 7) no amplification from human cell lines could be achieved. Lane 9 – DNA 100 bp ladder.
analyze the whole HRV2–HRV3 region (position 8–638) to reliably discriminate human cell lines. The detected polymorphisms were evenly distributed over the analyzed HRV1 region (Figure 4). Some cell lines differed only in one single polymorphism (Table 2). Thus it is essential to analyze the whole described sequence of HRV1 from position 15976 to 16497.
Sequencing results of HRV2 and HRV3 DNA sequencing of the HRV2–HRV3 region on 13 cell lines showed 13 distinct genotype sequences (Table 3). Therefore, sequences of the HRV2–HRV3 region also displayed specific polymorphisms (Table 3). Most frequently detected polymorphisms in this region were purine transition A-G at position 73 (9/13), 263 (13/13) and transversions at position 398 (T-A; 13/13), 410 (AT; 12/13) (Figure 5). Even though the highest number of polymorphisms was detected in the region of bp 100–199 of the HRV2–HRV3 locus (6/19), it is necessary to
Combined HRV1, HRV2 and HRV3 sequencing results Sequence informations achieved from the HRV1 or HRV3 could be combined to identify human cell lines more accurately as each cell line displayed specific polymorphisms. To discriminate cell lines by HRV2 sequencing data, those had to be combined with HRV1 or HRV3 data. The highest degree of resolution of unique genotypes was obtained for the HRV1 region.
Classification of the mtDNA polymorphisms found within HRV1, HRV2 and HRV3 Figure 6 shows the distribution of mtDNA polymorphisms. Neither insertion nor deletions were observed in the HRV regions. In contrast to HRV1, which showed a strong bias toward transitional changes (73 : 5), transi-
Table 1 Sequence of primers for amplification and sequencing of mitochondrial DNA Name
Origin
Locus
Sequence
Fragment size (bp)
Primer localization
mtReg-F1 mtReg-R1
Human Human
HRV1
50 -TCCACCATTAGCACCCAAAGC-30 50 -TCGGATACAGTTCACTTTAGC-30
521
L15976 H16497
mtReg-F2 mtReg-R2
Human Human
HRV2
50 -GGTCTATCACCCTATTAACCAC-30 50 -CTGTTAAAAGTGCATACCGCC-30
422
L8 H430
mtReg-F3 mtReg-R3
Human Human
HRV3
50 -CGCACCTACGTTCAATATTAC-30 50 -GGGTGATGTGAGCCCGTCTAA-30
541
L97 H638
mtmouse F1 mtmouse R1
Mouse Mouse
mHRV
50 -GGTTCTTACTTCAGGGCCATC-30 50 -TTGTTAATGTTTATTGCGTAA-30
417
15878 16295
mtmouse F2 mtmouse R2
Mouse Mouse
mHRV
50 -CCTTTCCTTCATACCTCAAAG-30 50 -AGCTTATATGCTTGGGGAAAA-30
442
15051 15493
F, forward primer; R, reverse primer. The position of primers is listed according to their 50 end location in the reference sequence for the human DNA (>gi|13959823|ref|NC_001807.3| Human mitochondrion, complete genome) and mouse DNA (>gi|342520|gb|J01420.1|MUSMTCG Mouse mitochondrion, complete genome). The Hematology Journal
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Figure 3 Results of PCR amplification of a mixed cell population (mouse cell line Baf3 and a human cell line WSU-NHL) with mouse-specific mtmouse1 (a: lanes 10–17), mtmouse2 (a: lanes 2–9) primers and the control human-specific mtreg 1 primers (b: lanes 1–8). Cells from a human cell line (WSU-NHL, 6 106 cells, lanes 2–9, 10–17 (panel a); lanes 2–8 (panel b) were mixed with different amount of cells from a mouse cell line (range from 1.32 106 to 1.32 101 cells; respectively, on lanes 8–3 and 16–11 (panel a). Lanes 9 and 17 on panel a contain amplification products from only mouse cells (Baf3; 1.32 106 cells). The mtmouse1 primers are more sensitive for the detection of a contamination compared to the mtmouse2 primers (detection limit: for mtmouse1 – 1.32 102 mouse cells; for mtmouse2 1.32 104 in 6 106 human cells). Lane a: 1 – DNA 100 bp ladder.
specific mtDNA sequence as highly contaminated with the Raji cell line. Another cell line labelled as the NIH3T3 cell line by the original investigator was characterized as a human cell line as a PCR product was only seen with humanspecific primers. By sequencing analysis, this cell line was identified as the 293T cell line.
Discussion
Figure 4 Percentage of polymorphic nucleotide positions in the HRV1 region. A, adenine; C, cytosine; G, guanine; T, thymidine.
tions and transversions were more evenly distributed in HRV2–HRV3 (P ¼ 0.0001). Similarly in HRV1, the majority of transitions were pyrimidine transitions (66/73), whereas the number of pyrimidine and purine transitions was identical in HRV2–HRV3 (29 to 29).
Identification of mislabelled cell lines Applying the above-described method on six cell lines obtained from other laboratories, three cell lines were identified as mislabelled. Two samples considered as transfected HL60 cell lines were characterized by the The Hematology Journal
One of the major problems in cell culture is the high prevalence of cross-contaminations, which is in the range of 10–30%1–3 and not sufficiently recognized in the general scientific community, namely, experimental data are still being published without documented authenticity of the cell lines, which may hamper the advances of cell-line-based research in scientific or biomedical area. Thus, the development of an easy, not labor-intensive method for cell line authentication is warranted. mtDNA analysis has been applied in several biomedical investigations of human evolution, for example, studies tracing the origin of modern humans17,18 or of certain human populations.19,20,21 In addition, mtDNA analysis is extremely effective in a forensic setting for the identification of criminals and victims of crimes or accidents.22,23 In previous studies, mtDNA polymorphisms were used in transfusion medicine and transplantation immunology to distinguish individuals in a
Table 2 Polymorphism of the HRV1 region in 30 cell lines
Base- Polypair morposi- phism tion
C-T C-T T-C C-T T-C T-C G-A T-A C-T A-G T-C T-C A-C T-C C-T A-G C-T C-T C-T T-C C-T C-T C-A C-T A-T C-T C-T G-A C-T C-T C-T T-C T-C T-C T-C A-G T-C T-C T-C A-G T-G T-C A-T
1 5 1 1 2 5 2 1 2 1 1 1 1 2 1 1 1 5 5 1 1 2 1 1 1 1 2 1 3 2 2 1 2 4 1 1 2 5 1 1 1 2 1
C A P A N
D O H H 2
K 5 6 2
M O N O M A C 6
G R A N T A 5 1 9
H B L
K A R P A S 4 2 2
M V 4 1 1
N B 4
H L 6 0
H E L A
J U R K A T
P A T U 8 9 8 8S
R A J I
U 9 3 7
W S U N H L
M C F 7
SW 48
L O V O
2 9 3 T
K A S U M I
L 4 2 8
J V M 2
P A T U 8 9 8 8T
N C E B
M E C 1
P L B 9 8 5
T H P 1
R E C 1
H E P
+ +
+
+
+
+ + +
+ +
+
+
+
+
+
+
+ +
+
+ +
+ + +
+ +
+
+
+
+ + +
+ +
+
+
+
+ +
+
+
+
mtDNA and the authentication of cell lines G Hutter et al
16030 16069 16093 16108 16124 16126 16129 16137 16148 16171 16172 16173 16183 16189 16193 16218 16222 16223 16224 16245 16249 16256 16258 16262 16265 16266 16270 16274 16278 16294 16296 16304 16305 16311 16324 16343 16356 16362 16391 16399 16414 16444 16453
Number of polymorphisms (n=30)
+ +
+
+ + +
+ +
+ + +
+
+ + + +
+
+
+
+ + +
+ + +
+
+
+
+ + + +
+
+
+ +
+
+
+ + + +
+ +
65
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Polymorphisms are identified based on the human mtDNA sequence >gi|13959823|ref|NC_001807.3| Human mitochondrion, complete genome.
66
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Basepair position 73 146 150 152 185 195 199 228 263 295 320 323 398 410 458 464 479 491 499
Polymorphism A-G T-C C-T T-C G-A T-C T-C G-A A-G C-T T-C T-C T-A A-T C-T C-T T-C T-C C-T
Number of polymorphisms (n=13) 9 1 5 1 3 3 1 4 13 5 1 1 13 12 1 3 1 4 2
Number of nucleotide polymorphisms per cell line
LOVO
MCF-7
HBL
NB-4
L-428
+
+
+
JVM-2
MONOMAC-6
PLB-985
Raji
WSU-NHL
+
+
+
+
+ +
+
+
+
MV4-11
ReC-1
JURKAT
+
+
+
+
+ + +
+
+ +
+ +
+
+
+
+ + +
+ + +
+
+ +
+
+ + +
+
+ + +
+
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ + +
+ + +
+ +
+ +
+
+ +
+ +
+
+ +
+
+
+
+
+ 3
3
5
5
+ 9
8
5
9
6
Polymorphisms are identified based on the human mtDNA sequence >gi|13959823|ref|NC_001807.3| Human mitochondrion, complete genome.
8
5
9
7
mtDNA and the authentication of cell lines G Hutter et al
Table 3 Polymorphism of the HRV2 and HRV3 HRV region in 13 cell lines
mtDNA and the authentication of cell lines G Hutter et al
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Figure 5 Percentage of polymorphic nucleotide positions in HRV2 and HRV3.
Figure 6 Number of nucleotide transitions/transversions in the HRV HRV1 (dark columns) and HRV2–HRV3 (light colored columns) region. Pyrimidine transitions (TC and CT) and purine transitions (AG and GA) were most frequently detected.
relatively homogenous population of Caucasian blood donors24,15 focusing on the HRV region of the control D-loop (HRV1–HRV3). This noncoding D-loop region contains various regulatory elements for replication and transcription11 and has a high variability based on a high mutation rate compared to nuclear DNA. This high variability is due to the limited availability of repair mechanisms in mitochondria.14,25,26,27
We therefore hypothesized that it is possible to discriminate and identify human LL cell lines and other human cell lines based on the polymorphisms of their HRV mtDNA regions (HRV1, HRV2, HRV3). By comparing the mtDNA sequences of the HRV regions with the published reference sequence (4gi|13959823|ref|NC_001807.3| Human mitochondrion, complete genome), it was possible to determine cell-line-specific polymorphisms of the HRV regions, which were evenly distributed over the HRV1, HRV2 and HRV3 regions. Nevertheless, the highest degree of discrimination could be achieved with the HRV1 sequences or the combined analysis of HRV2 and HRV3. Both approaches reliably discriminated cell lines by their unique mtDNA polymorphisms. Even though these regions are known to be highly variable, the cellline-specific polymorphisms remained unaltered after a high number of passages in cell culture (data not shown). To determine polymorphisms we compared the mtDNA sequences of the human cell lines to the reference sequence 4gi|13959823|ref|NC_001807.3| which slightly differs from the Cambridge reference sequence.9 Although the identified polymorphisms may differ if the Cambridge reference sequence is being applied, sequences unambiguously remain cell line specific. We therefore conclude that sequencing of the HRV regions of the human mtDNA can be applied for the discrimination and authentication of human cell lines. This easy and fast method will enable the establishment of a database of characterized polymorphisms as an additional instrument to reliably identify cell lines and detect cross-contaminations. In this study, we additionally demonstrate the detection of interspecies crosscontaminations. While the described method does not allow the detection of low-level intraspecies crosscontamination, further modifications of the method may achieve a very high sensitivity. In conclusion, we believe that the application of polymorphisms in the HRV-regions of the mtDNA represents an efficient approach for the authentication of cell lines, thereby avoiding the frequent problem of cross-contamination of cell lines.
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