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Carcinogenesis Advance Access originally published online on December 12, 2005
Carcinogenesis 2006 27(3):499-507; doi:10.1093/carcin/bgi299
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Carcinogenesis vol.27 no.3 © Oxford University Press 2005; all rights reserved.

Decreased expression of the human stem cell marker, Rex-1 (zfp-42), in renal cell carcinoma

Jay D. Raman 1, Nigel P. Mongan 2, Limin Liu 2, Satish K. Tickoo 3, David M. Nanus 4, Douglas S. Scherr 1 and Lorraine J. Gudas 2, *

1 Department of Urology, 2 Department of Pharmacology, 3 Department of Pathology and 4 Division of Hematology and Medical Oncology, The New York-Presbyterian Hospital, Weill Medical College of Cornell University, New York, NY, USA

* To whom correspondence should be addressed at: Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA. Tel: +1 212 746-6250; Fax: +1 212 746 8858; Email: ljgudas{at}med.cornell.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Rex-1 (Zfp-42) gene encodes a zinc finger family transcription factor which is highly expressed in mouse and human embryonic stem cells. It is one of several gene markers used to identify human stem cells. While several organs are known to harbor adult human stem cells, the presence and distribution of stem cells in both the normal and neoplastic adult kidney remains largely unknown. In this study we evaluated Rex-1 mRNA and protein expression in normal and malignant kidney tissue specimens from human patients. Rex-1 mRNA expression was determined using both reverse transcription and real-time PCR. REX1 protein expression was assessed by western analysis and immunohistochemistry, using an affinity-purified, polyclonal antibody to the REX1 protein. We found that 14 of 15 (93%) non-tumor renal parenchymal specimens demonstrated Rex-1 mRNA, compared with 5 of 14 (36%) renal tumors (P < 0.005). REX1 protein expression was detected in 21 of 23 (91%) non-tumor and in 7 of 19 (37%) tumor specimens (P < 0.001). Furthermore, in six of these seven renal tumor specimens where REX1 protein expression was detected, the levels were at least 3-fold lower than those in adjacent, normal kidney tissue. There were no differences in Rex-1 mRNA or protein expression among the various histologic subtypes of renal tumors (clear cell carcinoma, papillary carcinoma, chromophobe carcinoma and oncocytoma). Immunohistochemical staining confirmed the absence of REX1 in three renal tumor specimens (two clear cell and one papillary carcinoma), while the REX1 protein was detected in a small percentage of proximal tubular cells in normal renal tissue. Immunohistochemical staining of another stem cell marker, OCT4, demonstrated a similar pattern of protein expression in a small percentage of normal renal proximal tubular cells. In summary, we were able to detect Rex-1 mRNA and protein expression in over 90% of normal renal parenchymal specimens and we observed a significant reduction in REX1 expression in renal tumor specimens of all histologic subtypes.

Abbreviations: CSC, cancer stem cell; RCC, renal cell carcinoma; YY1, Ying-Yang1


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Tissue-specific stem cells exist in most adult organs of the body and can be defined as cells that have the capacity for self-renewal, as well as limited potential to differentiate into distinct cell lineages (1). Stem cells have been well documented in several organs (2), notably the hematopoetic system (3,4), breast (5), skin (6), intestinal epithelia (7) and neural tissues (8). Putative stem cell populations have also been identified in organs with less rapid rates of cell turnover, including the breast (5,9) and prostate (10). It is likely that the adult kidney possesses a population of organ-specific stem cells (11,12). Injury and repair models with renal regeneration have suggested several potential sites of progenitor cells within the kidney including the endothelium, mesangium and tubules (13). Other recent work with bromodeoxyuridine (BrdU) staining has indicated that the renal papilla may be a specific niche for a population of adult kidney stem cells (14,15). Despite these observations, the identification and location of such stem cells in the human kidney remain somewhat unclear.

Characterization of adult stem cells has been complicated partly by their low prevalence in tissue samples and cell culture systems. Recent success in identifying gene markers has facilitated the characterization of stem cell populations. Several genes, including Oct-4, Cripto, Sox2, Lefty A, Thy1, FGF4 and Rex-1 (zfp-42) have been identified as potential markers of human embryonic and adult stem cells (16–19). We first cloned Rex-1 from mouse F9 teratocarcinoma cells and identified it as a gene encoding a zinc finger family transcription factor exhibiting reduced expression in the presence of retinoic acid (20,21). Expression of Rex-1 has subsequently been detected in both murine (22,23) and human embryonic stem (ES) cells (24), as well as in murine differentiated spermatocytes (21). Rex-1 also plays a role in stem cell differentiation (25). Further work has demonstrated Rex-1 expression in several multipotent adult progenitor cells isolated from bone marrow, muscle and brain (26,27).

A defining characteristic of cancer cells is the ability to proliferate indefinitely (28). The similarity between the self-renewal mechanisms of stem cells and cancer cells has fostered the concept of cancer stem cells (CSCs) (29). It is possible that the presence and relative concentration of such CSCs may influence the malignant potential and future therapeutic options for human cancers (30). Renal cell carcinoma (RCC) affects nearly 35 000 people each year and is responsible for almost 12 000 deaths annually in the United States (31). RCC is a heterogeneous disease classified into various subtypes based on morphologic features (32). Clear cell RCC is the most common adult renal neoplasm, accounting for ~80% of kidney cancers. Less common subtypes of RCC include papillary (10–15%), chromophobe (5%), collecting duct (<1%) and unclassified (<2%) (33). Renal oncocytoma is an uncommon benign tumor which often resembles RCC clinically and pathologically (34). Recent cytogenetic studies have confirmed characteristic genetic alterations associated with each tumor type (35–37). These genetic variations may in part contribute to the variable clinical response to therapy when histologic subtypes are compared stage for stage. The stem cell hypothesis of carcinogenesis may also be relevant in terms of distinguishing the intrinsic behavior of different subtypes of RCC. Indeed, the distinct phenotypes of different renal-tumor types may reflect the influence of the local organ micro-environment on the tumor originating, transformed, adult stem cell (19). While a role for stem cells has been demonstrated for cancers of the hematopoietic system, breast and brain (38), the presence and distribution of stem cells in both the normal and neoplastic adult kidney remain largely unknown.

In this study we evaluated the mRNA and protein expression of the stem cell marker, Rex-1, in normal and malignant human renal tissue specimens. We further analyzed the pattern of REX1 protein expression using immunohistochemical techniques. In addition, the mRNA and protein expression of a second stem cell marker, OCT4, was evaluated for comparison. Finally, we examined different histologic subtypes of renal tumors to assess differences in Rex-1 expression.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patient tissue collection
We obtained 42 kidney specimens from 21 consecutive patients treated by either partial or radical nephrectomy for renal tumors at the New York-Presbyterian Hospital—Weill Cornell Medical Center between July 2004 and November 2004.

In 17 patients, samples were obtained from the visible tumor as well as from grossly uninvolved, adjacent renal parenchyma. Two of these 17 patients had two different areas of tumor and adjacent renal parenchyma sampled to assess for consistency within the same specimen. Four additional renal parenchymal specimens were obtained from the remaining four patients. These four specimens were procured from the opposite pole of any renal tumors and are considered to be representative of normal renal tissue. Twenty-nine of the 42 tissue specimens were equally divided such that western blot analysis and RT–PCR could be performed in parallel. For the samples processed immediately, seven tumor samples and seven non-tumor samples had frozen sections examined by pathology to obtain a diagnosis. All tumor samples were corroborated to be tumor on frozen sections and final pathology. The remaining samples that had no frozen sections were confirmed in the final pathologic reports. Namely, the reports state that a biopsy was taken from an area of tumor, or from an area of histologically normal kidney. The Institutional Review Board (IRB) of the New York-Presbyterian Hospital approved tissue procurement. Patient and tumor demographics are listed in Table I. Tumors were classified and staged based on final reports on the tissues submitted to the Department of Pathology, according to the 1997 American Joint Committee on Cancer—Union International Contre le Cancer (AJCC-UICC) Tumor-Node-Metastasis (TNM) classification (39).


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  		Table I. Demographics of patients and tumor characteristics of partial or radical nephrectomy specimens included in the RT–PCR and western blot analyses for Rex-1

 
RNA isolation and semiquantitative RT–PCR
Twenty-nine patient tissue specimens were procured and immediately placed into RNAlater (Ambion, Austin, TX). Six samples were initially washed with saline and showed no difference in RNA and protein expression of Rex-1 as compared with all of the remaining samples, making blood contamination an unlikely source of REX1 protein. Samples were then ground using a mortar and pestle in the presence of the TRIzol reagent (Invitrogen, Carlsbad, CA). First strand cDNA was synthesized from 3 µg of total RNA by reverse transcription with Superscript III Reverse Transcriptase (Invitrogen) at 42°C for 60 min and the synthesized cDNA was diluted to 200 µl with sterile, ultra pure water. Oligonucleotide primers were designed to amplify the Rex-1, Oct-4 and GAPDH cDNA products. Primers were designed to span intron–exon boundaries, thus preventing amplification of any contaminating genomic DNA. The Rex-1 primers (GenBank accession no. NM_174900 [GenBank] ) 5'-gctgaccaccagcacactaggc-3' (forward) and 5'-tttctggtgtcttgtctttgcccg-3' (reverse) generated a 298 bp product and the Oct-4 primers (GenBank accession no. NM_002701 [GenBank] ) 5'-gaccatctgccgctttgaggctctg-3' (forward) and 5'-gcgccggttacagaaccacactcgg-3' (reverse) generated a 301 bp product. A 472 bp fragment of GAPDH was generated using the sense primer 5'-agccacatcgctcagacac-3' and the antisense primer 5'-gaggcattgctgatgatcttg-3'.These GAPDH primer pairs were designed to have low homology with the 17 known GAPDH pseudogenes (40). PCR amplification of GAPDH was performed to confirm the integrity of cDNA. All primer pairs were evaluated using in vitro PCR software analysis (http://genome.ucsc.edu/). As Oct4 is now known to possess multiple processed pseudogenes (41), RNA which had not been reverse transcribed was used as a control to detect any contaminating genomic DNA in the cDNA preparation (Figure 2). Conditions for the PCRs on the patient samples consisted of 95°C for 5 min followed by cycles of 94°C for 30 s, primer annealing at 56–63°C for 30 s and template extension at 72°C for 45 s for 30–40 cycles as indicated in the figure legends. Each PCR contained ~40 ng of each oligonucleotide primer, 2 µl of cDNA, 2.5 x 10–2 U Taq polymerase and accompanying 1x buffer (Invitrogen), 1.5 mM MgCl2 and 0.2 mM deoxynucleoside triphosphates. Negative control PCRs using reverse-osmosis grade water in the place of template were incorporated in every PCR experiment. PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. The identity of the DNA product was confirmed by comparison of the PCR-amplified DNA to the predicted fragment size, as well as automated sequencing of the PCR product and comparison with the known DNA sequences of the target gene. PCR experiments were repeated at least three times on the patient samples with similar results.

Real-time PCR
Real-time PCR analysis was performed using a Bio-Rad MyIQ real time PCR system (Bio-Rad, Hercules, CA) and IQ SYBR green supermix (Bio-Rad). The threshold cycle (CT) was calculated by the MyIQ software. The oligonucleotide primer pairs for Rex-1 were, 5'-gaagaggccttcactctagtagtg-3' and 5'-tttctggtgtcttgtctttgcccg-3'; and for HPRT 5'-tgctcgagatgtgatgaagg-3' and 5'-tcccctgttgactggtcatt-3' as previously described (42). The CT values showed linear correlation with relative cDNA input for both the Rex-1 and HPRT primer pairs employed. Reaction conditions consisted of 95°C for 3 min to activate the polymerase followed by 45 cycles of 94°C for 10 s, primer annealing at 55°C for 10 s and extension at 72°C for 30 s; fluorescence was read after each cycle at 80°C. A standard curve was constructed for both Rex-1 and HPRT using cDNA generated from 5 µg RNA isolated from an N-tera2 human teratocarcinoma cell line, which expresses hRex-1 (N.P.Mongan, K.M.Martin and L.J.Gudas, manuscript in preparation). Expression of hRex1 was calculated in the unknown samples using the Genex Microsoft Excel macro (Bio-Rad). Real-time analysis was performed in triplicate for each cDNA sample with similar results. Negative control PCRs using water in place of template were performed.

Generation of human REX1 antibody
A C-terminal peptide of human REX1 (-SNNLKAHILTHANTNKNEQEGK-) was custom synthesized (Invitrogen). This peptide is located beyond the zinc finger domains, at the extreme C-terminus of the REX1 protein. The peptide was coupled to keyhole limpet hemocyanin and 100 µg (1 mg/ml solution) was repeatedly injected into rabbits and guinea pigs, using standard methods, to generate polyclonal antisera to the human REX1 peptide (Pocono Rabbit Farm and Laboratory, Canadensis, PA). The IgG fraction was then purified by DEAE-Affigel blue (Sigma, St Louis, MO) column chromatography. This was followed by affinity chromatography of the IgG fraction on a peptide affinity column made by coupling the REX1 peptide to cyanogen-bromide activated sepharose. The two-step purification steps were monitored by ‘dot-blot’ assays, with peptide spotted onto nitrocellulose discs. After incubation with column fractions, the discs were processed as described for the western blots described below.

Protein extraction and western blot analysis
Forty-two kidney tissue specimens from patients were procured and immediately placed into 2x sample buffer (100 mM Tris–HCl pH 6.8, 4% SDS and 20% glycerol). Samples were ground using a mortar and pestle, boiled for 5 min and the supernatant was stored at –20°C. Aliquots (100 µg) of whole cell lysate were separated on a 10% SDS–acrylamide gel and proteins transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were treated with a 1:25 dilution of the rabbit anti-human REX1 antibody overnight at 4°C and a 1:10 000 dilution of the secondary goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugated antibody (#sc-2030, Santa Cruz Biotechnology, Santa Cruz, CA), equivalent to 400 ng of the secondary antibody, for 1 h at room temperature. All dilutions were in phosphate-buffered saline containing 5% Blotto (Santa Cruz) and 0.1% Tween-20. Results were visualized by enhanced chemiluminescence reaction using the ECL SuperSignal (Pierce, Rockford, IL) and exposure to autoradiography. Goat anti-human GAPDH antibody (#sc-20357, Santa Cruz) at a dilution of 1:1000 was used to ensure consistent loading of protein extracts. Western blot experiments were repeated at least three times on the patient samples with similar results. Intensity of the western blot bands was quantified using NIH Image (version 1.63, National Institute of Health, Washington, DC).

COS cells (2 x 106), transiently transfected with a construct expressing hRex-1, were employed as a positive control in the western blot analyses. The Rex-1 expression plasmid was constructed by PCR amplification of the full-length protein-coding region of Rex-1 using Pfx proofreading polymerase (Invitrogen) from a Rex-1 EST (ATCC #5173608). Oligonucleotides, based on the Rex-1 protein coding sequence (AF450454 [GenBank] ), were designed to contain restriction sites (forward, BamH1: 5'-cgcggatccatgagccagcaactgaagaaacggg-3' and reverse, BglII: 5'-gaagatcttccaatgaggcatgtttgtcactgatc-3'). The amplified product was digested with BamH1 and BglII (New England BioLabs, Beverly, MA) and ligated into similarly digested pSG5 vector (Stratagene, Cedar Creek, TX). The sequence of the entire Rex-1 coding region in pSG5 was confirmed by direct automated sequencing and compared with the wild-type sequence. No mutations appeared to be introduced during PCR amplification.

Immunohistochemistry for REX1
Paraffin-embedded tissue sections from two patients and frozen sections from another patient were obtained. All three patients had Rex-1 expression confirmed by both RT–PCR and western blot analyses and thus were considered good candidates for attempted identification and localization of the REX1 protein by immunohistochemistry.

Paraffin-embedded sections
Five micrometer tissue sections were cut from the patient blocks and the sections were deparaffinized in Histo-Clear (National Diagnostics, Atlanta, GA) followed by rehydration in a graded series of ethanol. Antigen retrieval was performed by heat with the Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) in a pressure cooker for 8 min. A 3% solution of H2O2 was used to quench the endogenous peroxidase activity (15 min incubation). Slides were initially blocked with 1.5% goat serum for 30 min. This was followed by incubation with the affinity-purified, polyclonal rabbit anti-human REX1 primary antibody diluted 1:5 in 1.5% goat serum for 1 h at room temperature and then with 100 µl of HRP conjugated goat anti-rabbit secondary antibody (SuperPicture, Zymed, San Francisco, CA) for 30 min at room temperature. Color was developed with the 3,3'-diaminobenzidine chromogen substrate, followed by counterstaining with hematoxylin (Vector). The negative control normal and tumor sections were treated identically to all other sections, with the exception that 1.5% normal goat serum was used in place of the primary antibody. Primary antibody incubation was also performed in the presence of REX1 free peptide (1 µg/µl) to assess for non-specific primary antibody binding. All samples were analyzed using this identical protocol with the same reagents. The staining procedures were repeated at least three times with similar results.

Frozen sections
Seven micrometer tissue sections were cut from the patient's block and were stored at –80°C until staining. Slides were thawed at room temperature for 30 min and were fixed in 100% acetone for 2 min. Slides were initially blocked with 10% goat serum for 20 min at room temperature. This was followed by incubation with the affinity-purified, polyclonal rabbit anti-human REX1 primary antibody diluted 1:5 in 1% goat serum overnight at 4°C. The next day the slides were incubated with 100 µl of HRP conjugated goat anti-rabbit secondary antibody (SuperPicture, Zymed) for 30 min at room temperature. Endogenous peroxidase activity was quenched with a 0.3% solution of H2O2 for 20 min. Color was developed with the 3,3'-diaminobenzidine chromogen substrate, followed by counterstaining with hematoxylin (Vector). The negative control normal and tumor sections were treated identically to all other sections, with the exception that 1% goat serum was used in place of the primary antibody. The staining procedures were repeated at least three times with similar results.

Immunohistochemistry for OCT4
Paraffin-embedded sections from two patients with clear cell carcinoma were prepared as described above and staining was performed by methods previously described (43). Briefly, the sections were incubated overnight at 4°C with a 1:500 dilution of a commercially available polyclonal, goat anti-OCT4 antibody (#sc-8629, Santa Cruz). Subsequently, a 1:500 dilution of biotinylated rabbit anti-goat secondary antibody (#sc-2768, Santa Cruz) was applied to the sections and the bound antibody complex was visualized using the HRP avidin–biotin complex method.

Statistical analysis
Excel 2000 (Microsoft, Redmond, WA) software and SAS for Windows, version 9.1 (SAS Institute, Cary, NC) were used to perform all statistical calculations with P < 0.05 considered statistically significant. The chi-square ({chi}2) test was used to compare mRNA and protein expression in the normal and tumor tissue specimens.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
RT–PCR analysis of Rex-1 and Oct-4 expression in normal renal tissue versus renal tumors
Twenty-nine kidney tissue specimens obtained from 15 patients were evaluated for Rex-1 mRNA expression (Table I). Fourteen of 15 patients had both tumor and adjacent non-tumor specimens obtained, while one patient had only normal appearing renal tissue procured.

Results from a representative semi-quantitative RT–PCR analysis are shown in Figure 1 and the Rex-1 expression in all 29-tissue samples is summarized in Table II. Under the conditions employed, Rex-1 mRNA expression was noted in 14/15 (93%) non-tumor renal tissue specimens compared with 5/14 (36%) renal tumor specimens (P < 0.005). There was no difference in Rex-1 expression between the oncocytoma renal tumors and any of the different histologic subtypes of RCC (Table IV). One patient with clear cell carcinoma had absent Rex-1 expression in both normal and tumor specimens. No independent correlation between Rex-1 expression and Fuhrman histologic grade, pathologic tumor stage, tumor location, or tumor size was noted (data not shown).


Figure 1
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  		Fig. 1. Expression of Rex-1 mRNA in renal tumors compared with adjacent, non-tumor renal parenchyma. Results from nine patients are shown. Pairs 1–5, non-tumor and adjacent clear cell carcinoma; Pairs 6 and 7, non-tumor and adjacent papillary carcinoma; Pair 8, non-tumor and adjacent chromophobe carcinoma and Pair 9, non-tumor and an adjacent oncocytoma renal tumor. Amplification with PCR primers specific for GAPDH confirmed the integrity of the cDNA used. PCR for Rex-1 and GAPDH expression consisted of 40 and 30 cycles, respectively. All PCRs were performed in triplicate with similar results. One experiment is shown.

 

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  		Table II. Summary of semi-quantitative RT–PCR for Rex-1 mRNA expression in kidney tissue specimens

 
The results from a representative RT–PCR analysis of the Oct-4 gene are shown in Figure 2. Under the experimental conditions employed, Oct-4 mRNA expression was noted in all non-tumor and renal tumor specimens evaluated and these data support the observations of Tai et al. (19). There were no qualitative differences in expression between non-tumor and tumor specimens or between different histologic subtypes of renal tumors.


Figure 2
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  		Fig. 2. Expression of Oct-4 mRNA in renal tumors compared with adjacent, non-tumor renal parenchyma. Results from five patients are shown. Pairs 1–3, non-tumor and adjacent clear cell carcinoma; Pairs 4 and 5, non-tumor and adjacent papillary carcinoma. Amplification with PCR primers specific for GAPDH confirmed the integrity of the cDNA used. As Oct4 is now known to possess multiple processed pseudogenes (41), RNA which had not been transcribed was used as a control to detect any contaminating genomic DNA in the cDNA preparation). PCR for Oct-4 and GAPDH expression both consisted of 30 cycles. All PCRs were performed in triplicate with similar results. One experiment is shown.

 
Real-time PCR analysis of Rex-1 mRNA expression in normal renal tissue and adjacent renal tumors
Real-time PCR for Rex-1 expression was performed on RNA isolated from matched tumor and adjacent, non-tumor renal tissue from seven patients. The distribution of specimens included two clear cell carcinomas, two papillary carcinomas, one chromophobe carcinoma and one oncocytoma tumor. Expression of Rex-1 mRNA was not detected in four tumor samples in relation to adjacent normal renal tissue, while two tumor samples had detectable but lower levels of expression compared with normal (Figure 3). These results are consistent with our observations with semi-quantitative PCR (Figure 1 and Table II).


Figure 3
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  		Fig. 3. Real-time PCR was performed on RNA isolated from matched tumor and adjacent, non-tumor renal tissue from six patients: two with clear cell carcinoma (Patients 1 and 2, here); two with papillary carcinoma (Patients 3 and 4); one with chromophobe carcinoma (Patient 5); and one with oncocytoma (Patient 6). The relative level of Rex-1 expression for each specimen was determined with reference to the internal HPRT control. Real-time analysis was performed in triplicate for each cDNA sample (* = Not detected). Expression is depicted as fold expression relative to adjacent, non-tumor renal samples and the mean data presented. Asterisk indicates hRex1 expression <1% of adjacent renal non-tumor sample. Bars = standard deviation. Negative control PCRs using water in place of template were performed for each experiment.

 
Western blot analysis of REX1 protein expression in normal renal parenchyma versus renal tumors
Forty-two kidney tissue specimens obtained from 21 patients were evaluated for REX1 protein expression (Table I). Thirty-eight of these specimens were from 19 pairs of tumor and adjacent non-tumor, while the remaining four specimens were unpaired, normal renal parenchyma.

Results from a representative western blot analysis are shown in Figure 4, with a summary of REX1 protein expression provided in Table III. Transiently transfected COS cells expressing the human REX1 protein were used as positive control. REX1 protein expression was noted in 21/23 (91%) normal renal tissue specimens compared with 7/19 (37%) renal tumor specimens (P < 0.001). All of the normal and renal tumor specimens which exhibited REX1 protein expression also exhibited Rex-1 mRNA expression (Figure 1). In six of the seven renal tumor specimens where REX1 protein expression was detected, the levels were at least 3-fold lower than that in adjacent normal kidney tissue. There was minimal variance in REX1 expression in two patients who had two different areas of tumor and adjacent renal parenchyma sampled (data not shown). Similar to the mRNA data, there was no difference in REX1 protein expression between oncocytomas and any of the different histologic subtypes of RCC (Table IV). Two patients with RCC (one clear cell and one unclassified) exhibited no REX1 expression in either normal or malignant renal tissue. There was no correlation between REX1 protein expression and histologic tumor grade or stage.


Figure 4
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  		Fig. 4. REX1 protein expression in renal tumors compared with adjacent, non-tumor renal tissue by western blot analysis. Results from six patients are shown. Pairs 1–3, non-tumor and adjacent clear cell carcinoma; Pair 4, non-tumor and adjacent papillary carcinoma; Pair 5, non-tumor and adjacent chromophobe carcinoma; and Pair 6, non-tumor and an adjacent oncocytoma renal tumor. COS cells transiently transfected with a vector expressing Rex-1 or an empty vector were used as positive and negative controls, respectively. GAPDH was used to confirm the integrity of proteins. Ten micrograms of COS cell protein extract (positive and negative control) and 100 µg of extracted protein for each patient sample was used. Products were separated on a 10% SDS–acrylamide gel. Molecular masses from protein molecular weight markers are indicated on the left. All western blots were performed in triplicate with similar results. One experiment is shown.

 

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  		Table III. Summary of western blot analyses for REX1 protein expression in patient tissue specimens

 

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  		Table IV. Summary of mRNA and western blot analyses of Rex-1 expression stratified by histologic subtype of renal tumors

 
Immunohistochemical localization of REX1 and OCT4 protein expression in normal renal parenchyma and adjacent carcinoma
We obtained paraffin-embedded sections from two patients (with clear cell carcinoma) and frozen sections from another patient (with papillary carcinoma) whose RT-PCR and western blot analyses of tissue specimens revealed Rex-1 expression in normal renal parenchyma. Slides were stained to determine the cells in which REX1 was expressed (Figure 5). We found that the normal renal parenchyma exhibited granular staining of predominantly the cytoplasmic region of a small percentage (~ 1-2%) of proximal renal tubular cells (Figure 5A, E and I). In general, the staining was observed in most of the epithelial cells in a single tubule, but only a small percentage of tubules were stained (~1–2%). This staining was reproducible and was distinct from background staining (Figure 5C, G and K). We were also able to eliminate this staining by incubating with 1 µg/µL of free human REX1 peptide, thus demonstrating the specificity of binding (data not shown). We failed to identify REX1 immunostaining in any of the three carcinoma specimens (Figure 5B, F and J) or respective negative controls (Figure 5D, H and L).


Figure 5
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  		Fig. 5. Immunohistochemistry of REX1 expression in formalin-fixed, paraffin-embedded sections and frozen sections from human renal specimens. Three patient samples with renal cell carcinoma and adjacent, non-tumor renal parenchyma were stained with the affinity-purified, polyclonal rabbit anti-human REX1 antibody. The two formalin-fixed sections were from clear cell carcinoma specimens and the frozen section was from a papillary carcinoma specimen. (A–D) and (E–H) are from the two patients with clear cell carcinoma and panels I–L from the patient with papillary carcinoma. In normal renal tissue, the REX1 antibody predominantly stained the cytoplasmic region of a small percentage (~1–2%) of epithelial cells of the proximal convoluted tubules (A, E and I). This staining was not observed in negative controls (C, G and K). There was an absence of REX1 immunostaining in all three carcinoma specimens (B, F and J) and respective negative control (D, H and L). x600 magnification.

 
Immunohistochemistry for the OCT4 protein was performed on paraffin-embedded sections obtained from two patients with clear cell carcinoma. Similar to the REX1 staining, the normal renal tissues exhibited granular staining of predominantly the cytoplasmic region of a small percentage of proximal tubular cells (Figure 6A and C). We did not identify any OCT4 staining in either of the clear cell carcinoma specimens (Figure 6B and D) or the negative control (Figure 6E).


Figure 6
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  		Fig. 6. Immunohistochemistry of OCT4 expression in formalin-fixed, paraffin-embedded sections from two human clear cell carcinoma specimens. Staining was performed by techniques described in the Materials and methods section. In normal renal tissue, the OCT4 antibody predominantly stained the cytoplasmic region of a small percentage (~1–2%) of epithelial cells of the proximal convoluted tubules (A and C). This staining was not observed in the clear cell carcinoma specimens (B and D) or the negative control (E). x600 magnification.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The identification of stem cell populations in tissues with low regenerative potential has fostered interest in renal stem cells. The identification, isolation and regulation of such stem cells may provide innovative therapeutic options for acute renal failure, end stage renal disease and renal transplantation (11,44–46). It appears that kidney stem cells may exist in both renal tissue (12), as well as in the peripheral circulation. In a murine renal transplantation model, Poulsom et al. (47) utilized in situ hybridization to demonstrate that circulating stem cells can engraft in the kidney and differentiate into renal parenchymal cells. Other human and rodent experimental studies have suggested that bone marrow derived mesenchymal stem cells have the capacity to participate in renal repair in injury states (48–52). In these injury and regeneration models, it is possible that extrarenal stem cells are preferentially recruited to the kidney, thus confounding identification of native renal stem cells. It is also likely that organ specific renal stem cells do exist (12). BrdU staining can be used to identify ‘label retaining cells’ by virtue of the fact that these organ-specific stem cells exhibit limited cell divisions (53). This technique has identified diffuse labeling in the proximal, distal and collecting tubules, as well as in the renal papilla in normal kidneys suggesting the presence of a persistent renewing stem cell population (14,15,54). In addition, isolation of epithelial cells from renal tubules of normal adult rabbit kidneys has demonstrated the capacity for self-renewal, as well as for differentiation into renal tubular structures in vitro (55).

The utilization of stem cell molecular markers can facilitate the identification of organ-specific stem cells in adult tissues. As is the case for Rex1, expression of Oct4 is believed to be limited to and characteristic of stem cells (17). Indeed expression of Oct4 coincides with Rex1 expression in the multipotent progenitor cells thus far shown to express Rex1 (56,57). Tai et al. (19) have previously demonstrated Oct-4 gene expression in human adult stem cells, immortalized non-tumorigenic cells and tumor cells. They further identified Oct-4 expression in several adult human tissue derived stem cell cultures, including renal epithelial stem cells. Two other groups have recently shown that prostatic stem cells can be purified from isolated proximal duct regions by virtue of their high expression of the cell surface protein stem cell antigen 1 (Sca-1) and that Sca-1-enriched prostate-regenerating cells possess multiple stem cell properties that can initiate prostate tumorigenesis (58,59). We have recently observed Rex-1 expression in normal human epithelial cells and carcinoma cells in culture (N.P.Mongan et al. manuscript in preparation).

In this study, we investigated the expression of the human stem cell marker, Rex-1, in normal and neoplastic adult kidney tissue specimens. We demonstrated a high level of Rex-1 mRNA and protein expression in over 90% of normal renal parenchymal specimens. In addition, we showed that Rex-1 mRNA and protein expression decreased by greater than 3-fold in most of the renal tumor specimens of all histological subtypes. We were able to localize the REX1 protein predominantly to the cytoplasmic region of ~1–2% of proximal renal epithelial tubules in normal renal tissue, with staining absent in three RCC specimens. To better confirm our observations, we also assessed the expression of a second stem cell gene marker, Oct-4. We detected similar levels of the Oct-4 mRNA message in both the non-tumor and tumor specimens, but were only able to detect OCT4 protein expression by immunohistochemistry in normal renal tissue. Indeed, the staining for REX1 and OCT4 were very similar in that both stained a small percentage of proximal renal tubule epithelial cells (~1–2%), without any detectable expression in carcinoma specimens. This finding has important implications in normal kidney organogenesis and renal regeneration following injury. This may suggest a limited number of tubules, or stem cell niches, retain the capacity to participate in organ renewal and regeneration; or that a limited number of renal stem cell tubule niches (60) are locally activated at any given time.

Proximal renal tubule epithelium is known for its capacity for regeneration in the setting of toxic or ischemic injury (61–63). While hematopoietic stem cells may contribute to this regeneration (64), our data corroborates other published studies indicating that a population of renal stem cells is localized to the renal tubules (54). Further, the percentage of cells that express the REX1 protein is consistent with published reports of stem cell prevalence in other human organ systems (65,66). Given that REX1 is a transcription factor, we might have expected that the staining would predominantly be nuclear, as opposed to cytoplasmic. This, however, does not appear to be the case. We have previously described that REX1 is related to the Ying-Yang1 (YY1) transcription factor (25) (N.P.Mongan et al., manuscript in preparation). Recently, Palko et al. (67) demonstrated that the subcellular distribution pattern of YY1 altered during the cell cycle. Specifically, they report that YY1 is predominantly cytoplasmic at the G1 phase, primarily nuclear during the early and middle S phase and subsequently returns to the cytoplasm later in S phase. These findings suggest that YY1 and perhaps REX1 as well, localizes to the cytoplasm during most of the cell cycle. While the REX1 staining was observed in only a small percentage of proximal renal tubules (~1–2%), it appeared to involve most of the epithelial cells in a single tubule rather than the individual cells in a tubule. One explanation is that some proximal tubules may possess a greater stem cell population than others. Further, while these proximal tubules may appear separate, distinct and unrelated, these tubules may associate within a local, specialized spatial arrangement which constitutes a renal stem cell niche, analogous to that found in the epidermis (6). Another potential hypothesis is that following injury, stem cells contribute to the regeneration of renal tubular epithelial cells. It is possible that these newly regenerated tubular cells have yet to differentiate fully and thus still possess some stem cell markers, such as Rex-1.

The CSC hypothesis suggests that only a fraction of cells in the tumor possess the ability to proliferate and self-renew extensively. The remaining tumor cells lack this ability to proliferate and eventually evolve into a population of cells that becomes the tumor's phenotype (68). Given the similarity between ES cells and CSCs (69), it is postulated that embryonic gene markers may be a reasonable means to identify and isolate such CSCs. In our study, we found that the majority of renal tumor specimens demonstrated decreased mRNA and protein expression of the Rex-1 stem cell marker in comparison with normal adjacent renal parenchyma. There are several possible explanations for the apparent decrease in Rex1 expression in the majority of the tumor samples. It may indicate that these specific tumors possess fewer tumor initiating CSCs, relative to the total tumor mass, than tumors shown to express Rex1. Alternatively, it is possible that those renal tumors which lack hRex1 expression arose in a partially differentiated stem cell or consist primarily of moderately differentiated progeny of the originating CSC. When examining a second stem cell gene marker, Oct-4, we detected similar levels of the transcriptional message in tumor and non-tumor tissues, but noted an absence of OCT4 protein expression in tumor specimens. There are several potential explanations for these observations. One possibility is that the absence of detectable REX1 and OCT4 protein expression may indicate a lower prevalence, but not the absence of CSCs. Hence, PCR-based amplification may note gene expression (Rex-1, Figure 1; Oct-4, Figure 2), but the actual protein level may be below the threshold of detection. Another interesting possibility is that these tumors may have originated from a more differentiated, or partially committed, progenitor cell during the process of carcinogenesis (70,71). As a consequence, these tumors may lack some of the genes expressed in undifferentiated stem cells, such as Rex-1 (Figure 1), while expressing Oct-4 mRNA (Figure 2).

We detected Rex-1 expression in just over one-third of renal tumor specimens and the expression was always lower than that in non-tumor specimens. The prognostic significance of such expression in this fraction of renal tumors cannot be addressed in this study. The earlier detection of RCC has resulted in most newly diagnosed tumors being confined to the kidney (stages T1a and T1b) (72). As a consequence, it is not surprising that we were unable to correlate Rex-1 expression with either tumor pathologic stage or grade. Of particular interest will be the analysis of outcomes for patients with tumors of similar pathological stage and histological subtype with different levels of Rex-1 expression. Increased Rex-1 expression may suggest a higher concentration of CSCs within a particular tumor. A higher population of CSCs may portend a more aggressive phenotypic disease and may identify a sub-population of patients who would benefit from more vigilant screening or immediate adjuvant therapy. One therapeutic modality that has already been preliminarily explored in RCC is treatment with retinoids (73). Retinoids, which are known to decrease Rex-1 expression in mouse ES and F9 teratocarcinoma cells by promoting cellular differentiation (20), may be particularly beneficial in patients with tumors demonstrating high Rex-1 expression.

In summary, our data demonstrate high levels of expression of the human stem cell marker, REX1, in normal renal parenchyma, with localization to the proximal renal epithelial tubules. Furthermore, Rex-1 mRNA and protein expression is significantly decreased in most RCC specimens. Future studies are needed to better characterize the cellular role of REX1 and to establish the prognostic value of differential REX1 expression in renal tumors. We also plan to evaluate other putative stem cell gene markers in both normal and neoplastic adult and pediatric renal tissue.


   Acknowledgments
 
We thank the attending physicians in the Department of Urology and the peri-operative staff at the New York Presbyterian hospital for assistance in procuring tissue specimens. We thank the Gudas, Scherr and Nanus laboratories for scientific discussions and Karl Ecklund for editorial assistance. Grant support: This research was supported by NIH grants R01DE10389 and R01CA39036 (L.J.G.) and R01CA92542 (D.M.N.); and an American Association for Cancer Research—Cancer Research Foundation of America (AACR-CRFA) postdoctoral fellowship in cancer prevention (N.P.M.).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 13, 2005; revised November 8, 2005; accepted November 27, 2005.


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Isolation and Characterization of Kidney-Derived Stem Cells
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