approximately , 1.6 million new cases of lung cancer are diagnosed each year throughout the world .
since carcinogenesis is caused by the stepwise accumulation of genetic changes , it is very important to understand the alterations of chromosome and gene in cancer cells . the traditional giemsa banding ( g - banding ) technique is applied to detect cell sample with a higher mitotic index , but lung cancer cells have a lower mitotic index , which makes karyotypic analysis difficult . in our study , combining with the latest molecular biology techniques , we investigated the chromosomal and genetic alterations in the human lung adenocarcinoma cell line om . here
, we summarized the chromosomal and genetic abnormalities detected in lung cancer cells and discussed the possible implication of those alterations in the processes of tumorigenesis .
the lung adenocarcinoma cell line om was established in the second department of biology kochi medical school , kochi university , japan .
the cells in cell line om were from human lung adenocarcinoma tissues . after cultured and purified , these cells developed into a special cell line for cancer research .
the cultures were harvested and g - banding of the chromosomes was performed by hypo - osmolality , fixation , trypsinization , and giemsa ( invitrogen corp . , carlsbad , ca , usa ) staining .
samples were evaluated , and images were captured using an olympus bx60 fluorescence microscope ( olympus , tokyo , japan ) and analyzed with the aid of image analysis software ( cytovision , applied imaging corp . , santa clara , ca , usa ) .
after washing with ethanol , dried , concentrated , and purified , the sample and reference genomic dnas were digested and random primed labeled with cyanine-5/cyanine 3-dutp ( vysis corp .
cohybridization of these dnas to a comparative genomic hybridization ( cgh ) arrays was performed for 72 h at 37c .
, japan ) and data were extracted and analyzed using multiscan medical image analysis system ( sony corp . ,
tokyo , japan ) . extracted dna from a normal human was used as the internal control .
all the primers were provided by the invitrogen corp . , ( invitrogen corp . , carlsbad , ca , usa ) .
the amplified samples were analyzed by electrophoresis at 100 v on 1.5% agarose gels stained with ethidium bromide .
the visual of the bands of amplified dna were captured by a camera in the device .
, cary , nc , usa ) is useful statistical software to analyze this kind of data .
an exact 95% confidence interval ( ci ) using the clopper  pearson method was given for the positive rate of abnormal chromosomes and genes .
the lung adenocarcinoma cell line om was established in the second department of biology kochi medical school , kochi university , japan .
the cells in cell line om were from human lung adenocarcinoma tissues . after cultured and purified , these cells developed into a special cell line for cancer research .
the cultures were harvested and g - banding of the chromosomes was performed by hypo - osmolality , fixation , trypsinization , and giemsa ( invitrogen corp . , carlsbad , ca , usa ) staining .
the slides after denaturation were dehydrated in graded series of ethanol and air dried . the 24 xcyte mfish kit ( vysis corp . ,
samples were evaluated , and images were captured using an olympus bx60 fluorescence microscope ( olympus , tokyo , japan ) and analyzed with the aid of image analysis software ( cytovision , applied imaging corp . , santa clara , ca , usa ) .
dna extraction was performed with 2-propanol ( sigma - aldrich , japan ) . after washing with ethanol , dried , concentrated , and purified ,
the sample and reference genomic dnas were digested and random primed labeled with cyanine-5/cyanine 3-dutp ( vysis corp .
cohybridization of these dnas to a comparative genomic hybridization ( cgh ) arrays was performed for 72 h at 37c .
, japan ) and data were extracted and analyzed using multiscan medical image analysis system ( sony corp . ,
all the primers were provided by the invitrogen corp . , ( invitrogen corp . , carlsbad , ca , usa ) .
the amplified samples were analyzed by electrophoresis at 100 v on 1.5% agarose gels stained with ethidium bromide .
the visual of the bands of amplified dna were captured by a camera in the device .
, cary , nc , usa ) is useful statistical software to analyze this kind of data .
an exact 95% confidence interval ( ci ) using the clopper  pearson method was given for the positive rate of abnormal chromosomes and genes .
characterizations of complex chromosomal abnormalities in the human lung adenocarcinoma cell line om were showed by g - banding analysis , and the wrong number of chromosomes was present in almost all cells .
either aneuploid or polyploid karyotype was observed which displayed chromosome instability in the human lung adenocarcinoma cell line om .
g - banding showed chromosomal aberrations on all chromosomes except chromosome x ( 95% ci : 064% ) and 19 ( 95% ci : 064% ) [ figure 1 ] .
giemsa - banding analysis revealed chromosomal aberrations on all chromosomes except , 4 , 8 , 14 , 15 , 17 , and 19 .
the structurally abnormal chromosomes were analyzed by multiplex fluorescence in situ hybridization ( m - fish ) . as was illustrated in figure 2 ,
chromosome 10 is the most frequently ( 95% ci : 29100% ) involved in translocations with six different interchromosomal translocations , and its frequency was 46 times in each sample .
the other chromosomes involved in translocation were chromosomes 1 , 2 , 3 , 5 , 6 , 7 , 8 , 9 , 11 , 16 , and 18 , and the 95% ci minimum value of each statistical datum was more than 9% .
multiple numerical and structural aberrations were observed in the majority of cells from multiplex fluorescence in situ hybridization image .
the other chromosomes involved in translocation were chromosomes 2 , 5 , 9 , 11 , 18 and 3 , 21 .
cgh revealed that the gains in the region of chromosome bands 3q25.3 - 28 , 5p13 , 12q22 - 23.24 , and the losses in that of 3p25 - 26 , 6p25 , 6q26 - 27 , 7q34 - 36 , 8p22 - 23 , 9p21 - 24 , 10q25 - 26.3 , 12p13.31 - 13.33 , and 17p13.1 - 13.3 [ figure 3 ] .
array comparative genomic hybridization analysis showed partial deletion of in 3p24.1 - 26 , 6p25 , 6q25.2 - 27 , 7q32 - 36 , 8p22 - 23 , 9p21 - 24 , 10q25 - 26.3 , 12p13.31 - 13.33 , and 17p131 - 13.3 together with duplication at 3q24 - 28 , 5p13 , and 12q22 - 23.24 . according to the results obtained by cgh
the results of the polymerase chain reaction ( pcr ) experiments showed that the genes membrane metalloendopeptidase ( mme ) ( 95% ci : 188% ) , sucrase - isomaltase ( si ) ( 95% ci : 54100% ) , butyrylcholinesterase ( bche ) ( 95% ci : 16100% ) , and kininogen ( kng ) ( 95% ci : 172% ) were amplified in this region .
however , the amplification of genes histidine - rich glycoprotein ( hrg , 95% ci : 060% ) and mucin ( 95% ci : 052% ) were not observed [ figure 4 ] .
agarose gel electrophoresis of polymerase chain reaction amplification products from the human lung adenocarcinoma cell line om .
n : negative control ( normal human lung cell line ) ; om : human lung adenocarcinoma cell line om .
mme : membrane metalloendopeptidase ; si : sucrase - isomaltase ; bche : butyrylcholinesterase ; kng : kininogen ; hrg : histidine - rich glycoprotein ; muc : mucin .
characterizations of complex chromosomal abnormalities in the human lung adenocarcinoma cell line om were showed by g - banding analysis , and the wrong number of chromosomes was present in almost all cells .
either aneuploid or polyploid karyotype was observed which displayed chromosome instability in the human lung adenocarcinoma cell line om .
g - banding showed chromosomal aberrations on all chromosomes except chromosome x ( 95% ci : 064% ) and 19 ( 95% ci : 064% ) [ figure 1 ] .
giemsa - banding analysis revealed chromosomal aberrations on all chromosomes except , 4 , 8 , 14 , 15 , 17 , and 19 .
the structurally abnormal chromosomes were analyzed by multiplex fluorescence in situ hybridization ( m - fish ) . as was illustrated in figure 2 ,
chromosome 10 is the most frequently ( 95% ci : 29100% ) involved in translocations with six different interchromosomal translocations , and its frequency was 46 times in each sample .
the other chromosomes involved in translocation were chromosomes 1 , 2 , 3 , 5 , 6 , 7 , 8 , 9 , 11 , 16 , and 18 , and the 95% ci minimum value of each statistical datum was more than 9% .
multiple numerical and structural aberrations were observed in the majority of cells from multiplex fluorescence in situ hybridization image .
the other chromosomes involved in translocation were chromosomes 2 , 5 , 9 , 11 , 18 and 3 , 21 .
cgh revealed that the gains in the region of chromosome bands 3q25.3 - 28 , 5p13 , 12q22 - 23.24 , and the losses in that of 3p25 - 26 , 6p25 , 6q26 - 27 , 7q34 - 36 , 8p22 - 23 , 9p21 - 24 , 10q25 - 26.3 , 12p13.31 - 13.33 , and 17p13.1 - 13.3 [ figure 3 ] .
array comparative genomic hybridization analysis showed partial deletion of in 3p24.1 - 26 , 6p25 , 6q25.2 - 27 , 7q32 - 36 , 8p22 - 23 , 9p21 - 24 , 10q25 - 26.3 , 12p13.31 - 13.33 , and 17p131 - 13.3 together with duplication at 3q24 - 28 , 5p13 , and 12q22 - 23.24 .
according to the results obtained by cgh , gene analysis was performed in the 3q24 - 28 region .
the results of the polymerase chain reaction ( pcr ) experiments showed that the genes membrane metalloendopeptidase ( mme ) ( 95% ci : 188% ) , sucrase - isomaltase ( si ) ( 95% ci : 54100% ) , butyrylcholinesterase ( bche ) ( 95% ci : 16100% ) , and kininogen ( kng ) ( 95% ci : 172% ) were amplified in this region .
however , the amplification of genes histidine - rich glycoprotein ( hrg , 95% ci : 060% ) and mucin ( 95% ci : 052% ) were not observed [ figure 4 ] .
agarose gel electrophoresis of polymerase chain reaction amplification products from the human lung adenocarcinoma cell line om .
n : negative control ( normal human lung cell line ) ; om : human lung adenocarcinoma cell line om .
mme : membrane metalloendopeptidase ; si : sucrase - isomaltase ; bche : butyrylcholinesterase ; kng : kininogen ; hrg : histidine - rich glycoprotein ; muc : mucin .
lung cancer remains the leading cause of cancer - related morbidity and mortality worldwide . unlike in leukemia research
, lung cancer cell has a lower cell mitotic index , which makes karyotypic analysis difficult .
using modern genetic technologies , we investigated the chromosome and gene alterations in human lung adenocarcinoma of cell line om .
the goal of our study is to identify the alterations of chromosome and gene in human lung adenocarcinoma cell line om , which may provide new insights into the mechanisms of tumorigenesis . with high - resolution g - banding techniques , both the numerical and structural chromosomal abnormalities were observed .
g - banding analysis revealed that the changes in chromosome number were present in most cells , and either aneuploidy or polyploidy was identified within them .
in om cell line , certain chromosomes frequently participated the structural rearrangement , prominent among them being chromosome 3 , 6 , 7 , 9 , 10 , 11 , 18 , and y. although g - banding is a fundamental technique for cytogenetic study , it has limited utility in instances of cryptic or very complex rearrangements .
m - fish is a powerful technique that can be used for identifying uniquely all 24 chromosome types of the human genome .
the m - fish image showed that the highest frequency of translocation was in chromosome 10 . in genetics ,
a chromosome translocation is caused by rearrangement of parts between nonhomologous chromosomes , and the distinct classes of chromosomal rearrangements create proto - oncogenes .
loci on chromosome 10 include microseminoprotein beta , which encodes beta - microseminoprotein , a primary constituent of semen and a proposed prostate cancer biomarker .
moreover , multiple tumor - suppressive genes are located in chromosome 10 , and the translocations of chromosome 10 may lead to damage of these tumor - suppressive genes .
therefore , frequent translocations of chromosome 10 in cell line om may be catastrophic genomic events and play key roles in tumorigenesis . in general , regional gains on chromosome
indicate that oncogenes may exist in this region , while the chromosome losses have been interpreted as an evidence that the region has affected a certain tumor suppressor genes .
cgh analysis revealed that gain regions were observed on chromosomes 3q25.3 - 28 , 5p13 , and 12q22 - 23.24 , which indicated that there may exist oncogenes that were activated in the gain regions . while loss regions were detected in chromosome 3p25 - 26 , 6p25 ,
6q26 - 27 , 7q34 - 36 , 8p22 - 23 , 9p21 - 24 , 10q25 - 26.3 , 12p13.31 - 13.33 , and 17p13.1 - 13.3 , which indicated that some tumor suppressor genes may be located in these regions .
it is well - known that tumor suppressor genes p16 and p53 were located in chromosome 9p21 and 17p13 , respectively .
based on the above results , we detected amplification of genes at chromosome band 3q24 - 28 by pcr in the cell lines .
the genes bche , mme , kng , and si were amplified in the chromosomal region .
the bche gene provides instructions for making the pseudocholinesterase enzyme , which has a variety of physiological effects such as the metabolism of the cocaine and heroin and the breakdown of organophosphorus esters .
recent studies indicated that bche gene was amplified in the cells of lung carcinomas , which were consistent with our results .
observed the amplification of genes bche and slc2a2 at 3q26 in squamous cell lung carcinomas , whereas no amplification has been found in genes mme and kng .
mme gene is expressed in both normal and neoplastic cells as a common acute leukemia antigen .
in squamous cell lung carcinoma , the high mme gene expression was significantly associated with poor overall survival .
there were many studies on the expression of mme gene in lung cancer cells ; however , these results were quite conflicting .
many factors may be responsible for the difference among the results , such as experimental conditions , experimental methods , and pathological types of lung cancer .
the kng gene , which is one of the members of a cystatin - like superfamily , has potential roles in angiogenesis and ( or ) tumor development .
kinins that derived from kng involved in cell proliferation , leukocyte activation , cell migration , endothelial cell activation , and nociception .
thought that kng may be a novel therapeutic target in chronic lymphocytic leukemia and the possible association with prognosis .
we observed that kng gene was amplified in lung adenocarcinoma cell line om , which suggested that kng gene was likely to be involved in tumorigenesis of lung cancer cells .
although there are numerous studies elucidating the role of si gene in the digestion of dietary carbohydrates , there are limited data indicating a specific involvement of si gene in the genesis of human cancers . our experiments demonstrated that si gene was amplified on chromosome band 3q24 - 28 . since cancer is attributed to multi - genetic alterations accumulated in the cells , we speculated si gene may be involved in tumorigenesis.in summary , our findings suggested that most of those examined cells exhibited multiple complex karyotypes in human lung adenocarcinoma cell line om .
chromosome 10 was frequently involved in chromosomal translocation , which may be catastrophic genomic events and play key roles in tumorigenesis . according to the results of cgh
, we speculated that the oncogenes may be located at 3q25.3 - 28 , 5p13 , and 12q22 - 23.24 , while tumor suppressor genes may exist in 3p25 - 26 , 6p25 , 6q26 - 27 , 7q34 - 36 , 8p22 - 23 , 9p21 - 24 , 10q25 - 26.3 , 12p13.31 - 13.33 , and 17p13.1 - 13.3 . in human lung adenocarcinoma cell line
om , at least four genes ( mme , si , bche , and kng ) were involved in carcinogenesis .
 background : lung cancer has become the leading cause of death in many regions . 
 carcinogenesis is caused by the stepwise accumulation of genetic and chromosomal changes . 
 the aim of this study was to investigate the chromosome and gene alterations in the human lung adenocarcinoma cell line om.methods:we used giemsa banding and multiplex fluorescence in situ hybridization focusing on the human lung adenocarcinoma cell line om to analyze its chromosome alterations . 
 in addition , the gains and losses in the specific chromosome regions were identified by comparative genomic hybridization ( cgh ) and the amplifications of cancer - related genes were also detected by polymerase chain reaction ( pcr).results : we identified a large number of chromosomal numerical alterations on all chromosomes except chromosome x and 19 . 
 chromosome 10 is the most frequently involved in translocations with six different interchromosomal translocations . 
 cgh revealed the gains on chromosome regions of 3q25.3 - 28 , 5p13 , 12q22 - 23.24 , and the losses on 3p25 - 26 , 6p25 , 6q26 - 27 , 7q34 - 36 , 8p22 - 23 , 9p21 - 24 , 10q25 - 26.3 , 12p13.31 - 13.33 and 17p13.1 - 13.3 . 
 and pcr showed the amplification of genes : membrane metalloendopeptidase ( mme ) , sucrase - isomaltase ( si ) , butyrylcholinesterase ( bche ) , and kininogen ( kng).conclusions : the lung adenocarcinoma cell line om exhibited multiple complex karyotypes , and chromosome 10 was frequently involved in chromosomal translocation , which may play key roles in tumorigenesis . 
 we speculated that the oncogenes may be located at 3q25.3 - 28 , 5p13 , 12q22 - 23.24 , while tumor suppressor genes may exist in 3p25 - 26 , 6p25 , 6q26 - 27 , 7q34 - 36 , 8p22 - 23 , 9p21 - 24 , 10q25 - 26.3 , 12p13.31 - 13.33 , and 17p13.1 - 13.3 . 
 moreover , at least four genes ( mme , si , bche , and kng ) may be involved in the human lung adenocarcinoma cell line om . 