cervical carcinoma is the second most common cause of cancer - related deaths in women worldwide , with a global annual incidence of approximately 470,000 new cases and an estimated 233,000 deaths per year .
management for early cervical cancers generally involves radical abdominal hysterectomy and radiotherapy , with good results in terms of survival and quality of life .
however , the 5-year survival rate of advanced patients is from 3.2% to 13% , which is due to their resistance to the traditional treatments .
mounting evidence suggests that cancer may in fact arise from a transformed stem cell that is able to self - renew , differentiate into diverse progenies , and drive continuous growth .
aldh is a detoxifying enzyme that oxidizes intracellular aldehydes and confers resistance to alkylating agents [ 47 ] .
recent studies have shown that aldh1 is a cancer stem cell marker and that its presence strongly correlates with tumor malignancy and self - renewal properties of stem cells in different tumors , including breast cancer , hepatoma , colon and lung cancer [ 811 ] ] . however , whether aldh1 can be a useful marker of csc and/or aid cancer diagnosis in cervical carcinoma is still an open question .
given this evidence , we investigated whether cervical carcinomas contain a tumorigenic stem cell - like population .
the human cervical cancer cell lines c33a , caski , hela and siha were stored in our lab .
the cells were cultured in rpmi 1640 media ( invitrogen life technologies , carlsbad , ca ) supplemented with 10% fetal bovine serum and incubated at 37c in an atmosphere containing 5% co2 .
tumors from the uterine cervix were collected from archives of the department of gynecology at the 2nd affiliated hospital of sun yat - sen university .
a total of 112 specimens were analyzed , whose diagnoses were invasive squamous carcinoma ( 89 cases ) and adenocarcinoma ( 20 cases ) , and normal cervical epithelium ( 3 cases ) obtained from patients undergoing hysterectomy for various non - malignant causes served as normal controls .
formalin - fixed paraffin - embedded specimens were cut into 3m sections and mounted on glass slides pre - treated with poly - l - lysine .
sections were boiled for 15 minutes in citrate buffer ( ph 6 ) , cooled in the same buffer , and subsequently incubated for 10 minutes in 0.3% h2o2 to block the endogenous peroxidase activity .
aldh1 antibody ( santa cruz ) was used at a 1/100 dilution stored overnight at 4c in a humid chamber , and rinsed in pbs .
sections were washed and incubated with horseradish peroxidase ( hrp)-conjugated anti - mouse secondary antibody ( 1:100 ) ( zymed , san francisco , ca ) for 60 minutes at room temperature , followed by abc vectastain kit ( vector laboratories , burlingame , ca , usa ) for 5 minutes .
replacement of the monoclonal antibody with mouse igg1 protein of the same concentration was used as a negative control .
immunohistochemical staining of aldh1 was classified as 3 + ( 50% positive tumor cells ) , 2 + ( < 50% but 10% ) , 1 + ( < 10% ) , or negative ( 0% ) .
cd133 antibody ( ab19898 ) and smad3 antibody ( ab28379 ) were used at a 1/100 dilution with anti - rabbit secondary antibody ( ab6721 ) .
the population with high aldh enzymatic activity was isolated by use of the aldefluor kit ( stemcell technologies , durham , nc ) .
cells were suspended in aldefluor assay buffer containing aldh substrate ( baaa , 1 mol / l per 110 cells ) and incubated for 45 minutes at 37c to allow the conversion of aldefluor substrate , a green fluorescent product retained within the cell due to its negative charge .
for each experiment , an aliquot of aldefluor - stained cells was immediately quenched with 5 l 1.5 mm diethylaminobenzaldehyde ( deab ) , a specific aldh inhibitor , to serve as a negative control .
the sorting gates were established using as negative controls the cells stained with pi only ; for viability , the aldefluor - stained cells treated with deab ; and the staining with secondary antibody alone .
cells were blocked with fcr blocking reagent and stained with cd133 mouse igg1 antibody conjugated to phycoerythrin ( pe ) ( miltenyi biotec inc . , auburn , ca ) .
mouse igg1-pe ( bd biosciences , san jose , ca ) was the isotype control antibody .
all reactions were done in a 20 l reaction volume in triplicate by sybr green real - time pcr universal reagent ( genepharma co. , ltd . ) and mx-3000p real - time pcr machine ( stratagen ) .
standard curves were generated and the relative amount of aldh1 and cd133 were normalized to -actin ( 2 ) .
primers are aldh1 ( forward , 5-ctgctggcgacaatggagt-3 ; reverse , 5-gtcagcccaacctgcacag-3 ) , cd133 ( forward , 5-caaccctgaactgag gcagc-3 ; reverse , 5-ttgatagccctgttggaccag-3 ) and -actin ( forward , 5-gcatgggtcagaaggattcct-3 ; reverse , 5-tcgtcccagttggtgacgat-3 ) was used as endogenous control .
protein was extracted from cervical cancer cell lines c33a , caski , hela and siha .
briefly , 10 g protein of each sample was subjected to sds - page on 10% polyacrylamide gels , electrophoretically transferred to pvdf membranes ( millipore , billerica , ma , usa ) .
the membranes were blocked for 1 hour at room temperature , and then incubated overnight at 4c in tris buffered saline with 0.05% tween ( tbst ) containing 5% dry milk and detected with mouse monoclonal igg1 anti - aldh1(bd , usa ) , rabbit polyclonal igg anti - cd133 ( abcam , usa ) and commercial ecl kit ( pierce , rockford , il , usa ) .
protein loading was estimated using mouse anti--actin monoclonal antibody ( sigma , usa ) and peroxidase - conjugated goat anti - mouse igg ( multisciences biotech co. ltd ) .
the human cervical cancer cell lines c33a , caski , hela and siha were stored in our lab .
the cells were cultured in rpmi 1640 media ( invitrogen life technologies , carlsbad , ca ) supplemented with 10% fetal bovine serum and incubated at 37c in an atmosphere containing 5% co2 .
tumors from the uterine cervix were collected from archives of the department of gynecology at the 2nd affiliated hospital of sun yat - sen university .
a total of 112 specimens were analyzed , whose diagnoses were invasive squamous carcinoma ( 89 cases ) and adenocarcinoma ( 20 cases ) , and normal cervical epithelium ( 3 cases ) obtained from patients undergoing hysterectomy for various non - malignant causes served as normal controls .
formalin - fixed paraffin - embedded specimens were cut into 3m sections and mounted on glass slides pre - treated with poly - l - lysine .
sections were boiled for 15 minutes in citrate buffer ( ph 6 ) , cooled in the same buffer , and subsequently incubated for 10 minutes in 0.3% h2o2 to block the endogenous peroxidase activity . aldh1 antibody ( santa cruz ) was used at a 1/100 dilution stored overnight at 4c in a humid chamber , and rinsed in pbs .
sections were washed and incubated with horseradish peroxidase ( hrp)-conjugated anti - mouse secondary antibody ( 1:100 ) ( zymed , san francisco , ca ) for 60 minutes at room temperature , followed by abc vectastain kit ( vector laboratories , burlingame , ca , usa ) for 5 minutes .
replacement of the monoclonal antibody with mouse igg1 protein of the same concentration was used as a negative control .
immunohistochemical staining of aldh1 was classified as 3 + ( 50% positive tumor cells ) , 2 + ( < 50% but 10% ) , 1 + ( < 10% ) , or negative ( 0% ) .
cd133 antibody ( ab19898 ) and smad3 antibody ( ab28379 ) were used at a 1/100 dilution with anti - rabbit secondary antibody ( ab6721 ) .
the population with high aldh enzymatic activity was isolated by use of the aldefluor kit ( stemcell technologies , durham , nc ) .
cells were suspended in aldefluor assay buffer containing aldh substrate ( baaa , 1 mol / l per 110 cells ) and incubated for 45 minutes at 37c to allow the conversion of aldefluor substrate , a green fluorescent product retained within the cell due to its negative charge . for each experiment ,
an aliquot of aldefluor - stained cells was immediately quenched with 5 l 1.5 mm diethylaminobenzaldehyde ( deab ) , a specific aldh inhibitor , to serve as a negative control .
the sorting gates were established using as negative controls the cells stained with pi only ; for viability , the aldefluor - stained cells treated with deab ; and the staining with secondary antibody alone .
cells were blocked with fcr blocking reagent and stained with cd133 mouse igg1 antibody conjugated to phycoerythrin ( pe ) ( miltenyi biotec inc . , auburn , ca ) .
mouse igg1-pe ( bd biosciences , san jose , ca ) was the isotype control antibody .
all reactions were done in a 20 l reaction volume in triplicate by sybr green real - time pcr universal reagent ( genepharma co. , ltd . ) and mx-3000p real - time pcr machine ( stratagen ) .
standard curves were generated and the relative amount of aldh1 and cd133 were normalized to -actin ( 2 ) .
primers are aldh1 ( forward , 5-ctgctggcgacaatggagt-3 ; reverse , 5-gtcagcccaacctgcacag-3 ) , cd133 ( forward , 5-caaccctgaactgag gcagc-3 ; reverse , 5-ttgatagccctgttggaccag-3 ) and -actin ( forward , 5-gcatgggtcagaaggattcct-3 ; reverse , 5-tcgtcccagttggtgacgat-3 ) was used as endogenous control .
protein was extracted from cervical cancer cell lines c33a , caski , hela and siha .
briefly , 10 g protein of each sample was subjected to sds - page on 10% polyacrylamide gels , electrophoretically transferred to pvdf membranes ( millipore , billerica , ma , usa ) . the membranes were blocked for 1 hour at room temperature , and then incubated overnight at 4c in tris buffered saline with 0.05% tween ( tbst ) containing 5% dry milk and detected with mouse monoclonal igg1 anti - aldh1(bd , usa ) , rabbit polyclonal igg anti - cd133 ( abcam , usa ) and commercial ecl kit ( pierce , rockford , il , usa ) .
protein loading was estimated using mouse anti--actin monoclonal antibody ( sigma , usa ) and peroxidase - conjugated goat anti - mouse igg ( multisciences biotech co. ltd ) .
aldh1 expression was observed in 23/89 ( 89 cases ) cases of invasive squamous carcinoma and 4/20 cases of adenocarcinoma ( 20 cases ) ( figure 1a  d ) .
no aldh1 was expressed in normal cervical epithelium obtained from patients undergoing hysterectomy for various non - malignant causes .
the majority of positive staining demonstrated focal moderate staining , and occasionally moderate diffuse staining with moderate intensity . among positive staining cases in primary cervical carcinomas and metastatic carcinomas , staining tended to be diffuse , with a spectrum ranging from weak to moderate - to - strong .
the results of immunostaining of the tumor microarrays , organized according to clinicopathologic characteristics of the patients , are shown in table 1 .
correlation was found between the expression of the aldh1 and lymph nodal metastasis in cervical carcinoma , and patients with recurrent disease were more likely to have a higher aldh1 expression ( table 1 ) .
we also found the expression of aldh1 has some relationship with smad3 ( table 2 ) .
we utilized the aldefluor assay to assess the presence and size of the population with aldh enzymatic activity in normal human breast epithelium .
analysis of c33a , caski , hela and siha cells showed an average of 3.10.26% , 22.072.63% , 11.633.54% and 27.533.96% aldefluor - positive population , respectively ( figure 3a  f ) .
we also studied expression of cd133 by the cervical cancer cell lines c33a , caski , hela and siha .
figures 4a  d show the 0.220.01% , 0.670.03% , 0.430.06% and 3.781.02% cd133-positive populations , respectively ( the control antibody background was not subtracted because it did not overlap cd133 fluorescence ) .
aldh1 mrna expression was significantly higher in cervical cell lines than was cd133 mrna expression ( figure 5 ) .
aldh1 protein was detected as a single band at 55 kda in all of the cervical cell lines tested by western blot ( figure 6 ) .
aldh1 expression was observed in 23/89 ( 89 cases ) cases of invasive squamous carcinoma and 4/20 cases of adenocarcinoma ( 20 cases ) ( figure 1a  d ) .
no aldh1 was expressed in normal cervical epithelium obtained from patients undergoing hysterectomy for various non - malignant causes .
the majority of positive staining demonstrated focal moderate staining , and occasionally moderate diffuse staining with moderate intensity . among positive staining cases in primary cervical carcinomas and metastatic carcinomas , staining tended to be diffuse , with a spectrum ranging from weak to moderate - to - strong .
the results of immunostaining of the tumor microarrays , organized according to clinicopathologic characteristics of the patients , are shown in table 1 .
correlation was found between the expression of the aldh1 and lymph nodal metastasis in cervical carcinoma , and patients with recurrent disease were more likely to have a higher aldh1 expression ( table 1 ) .
we also found the expression of aldh1 has some relationship with smad3 ( table 2 ) .
we utilized the aldefluor assay to assess the presence and size of the population with aldh enzymatic activity in normal human breast epithelium .
analysis of c33a , caski , hela and siha cells showed an average of 3.10.26% , 22.072.63% , 11.633.54% and 27.533.96% aldefluor - positive population , respectively ( figure 3a  f ) .
we also studied expression of cd133 by the cervical cancer cell lines c33a , caski , hela and siha .
figures 4a  d show the 0.220.01% , 0.670.03% , 0.430.06% and 3.781.02% cd133-positive populations , respectively ( the control antibody background was not subtracted because it did not overlap cd133 fluorescence ) .
aldh1 mrna expression was significantly higher in cervical cell lines than was cd133 mrna expression ( figure 5 ) .
aldh1 protein was detected as a single band at 55 kda in all of the cervical cell lines tested by western blot ( figure 6 ) .
the cancer stem cell hypothesis supposes that the cancer stem cells may relate to the cancer risk assessment , early detection , prognostication and prevention .
the development of more effective cancer therapies   may require targeting cancer stem cell populations .
the success of these new approaches concentrates on the identification , isolation , and characterization of cancer stem cells .
the study of cervical cancer sc would be greatly enhanced by availability of specific markers to identify and isolate these cells .
the aldehyde dehydrogenase ( aldh ) families of enzymes are cytosolic isoenzymes that are responsible for oxidizing intracellular aldehydes and contributing to the oxidation of retinol to retinoic acid in early stem cell differentiation .
visus et al . suggested that aldh1a1 is a marker for distinguishing premalignant cells in head and neck squamous cell carcinoma ( hnscc ) , and the increased incidence of aldh1 expression correlated positively with the staging of patients .
bin chang et al . analyzed the associations between the expression of the aldh1 and clinical factors ( diagnosis , tumor grade , stage , and clinical response to chemotherapy ) , as well as overall and disease - free survival .
they found aldh1 was a favorable prognostic factor of ovarian carcinoma . in breast carcinomas , the expression of aldh1 detected by immunostaining was correlated with poor prognosis .
a key finding of our study was that immunohistochemistry identifies a small subpopulation of aldh1 + cells localized in the cervical carcinoma . in the current study , high levels of aldh1 expression
were observed in patients with recurrent or metastatic disease , suggesting that aldh1 might be a potential independent prognosis predictor in cervical cancer .
loss of responsiveness to tgf--induced cell growth inhibition during cancer development is a feature of many tumors , including cervical cancer . in this study
, we found that increased smad3 protein expression in cervical cancers was associated with aldh1 expression .
a second key finding of our study is that aldh - based markers can be used to track cervical sc during cervical tumorigenesis . utilizing in vitro assays ,
aldh activity in cells was measured by using dansyl aminoacetaldehyde , and the activity of aldh was used to isolate precursor cells . unlike the previously described breast cancer stem cell phenotype , which requires the use of a combination of 10 surface antigens , testing for aldh1 activity is a simple method for identifying normal and cancer stem cells .
cells with high aldh activity contain the tumorigenic cell fraction able to self - renew and to recapitulate the heterogeneity of the parental tumor .
these cells may also have the highest ability to grow in vivo in a xenotransplantation animal model .
we observed high levels of aldh1 in primary cultures obtained from resected tumor tissue , confirming that aldh1 overexpression is not a phenomenon restricted to established cell lines .
thus far cellular markers including cd133 have been used to identify cscs in several tumors .
recently , the relevance of cd133 as a reliable csc marker has been doubted , since it was shown that even cd133-negative gbm cells may behave as brain cscs . however , in cervical carcinoma , we found the expression of cd133 was very limited .
since aldh1 is also expressed in hematopoietic and neuronal stem cells , this marker may prove useful for the detection and isolation of cancer stem cells in other malignancies , thus facilitating the application of cancer stem cell biology to clinical practice .
our study suggests that aldh1 expression may be used to detect cervical cancer stem cells .
identification of aldh1 as a potential marker of cancer stem cells opens important new avenues of research in cervical carcinogenesis .
 summarybackgroundaldh1 has been shown to play a role in the early differentiation of stem cells in some human malignancies . 
 whether cancer stem cells occur in aldh1-associated cervical cancer is not known.material/methodswe tested the hypothesis that cervical carcinomas contain subpopulations of cells that express aldh1 . 
 the following sources of cervical carcinoma tissues were examined for the presence of stem cell marker aldh1 by immunohistochemistry . 
 flow cytometric isolation of cancer cells was based on enzymatic activity of aldh ( aldefluor assay ) . 
 the mrna and protein levels of aldh1 were investigated by qrt - pcr and western blot , respectively . 
 we also detected the expression of cd133 identified as a stem cell marker for several cancers.results23/89 samples of invasive squamous carcinoma and 4/20 samples of adenocarcinomas exhibited immunoreactivity to stem cell marker aldehyde dehydrogenase 1 ( aldh1 ) . 
 expression of aldh1 was found in 24.77% of the samples . 
 flow cytometric analysis , qrt - pcr and western blot also confirmed the presence of small subpopulations of aldh1-positive cells . 
 in contrast , we found cervical carcinoma had low cd133 
 population.conclusionscervical carcinoma contains a small subpopulation of cells that may relate to a cancer stem cell - like phenotype , aldh1 . 
Background
Material and Methods
Cell Culture
Tissue samples
Immunohistochemical staining
ALDEFLUOR assay and separation of the ALDH-positive population by FACS
Flow cytometry for CD133
Quantitative Real-Time PCR
Western blot
Results
ALDH1 expression and localization in cervical carcinoma tissues
The ALDEFLUOR-positive population isolated from cervical cell properties
ALDH1 mRNA expression
ALDH1 protein expression
Discussion
Conclusions