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Department of Biochemistry and Molecular Genetics [K. W. F., S. R., I. D. L., S. M. L-R., M. R. H., T. R. B., L. T. C., J. M. R.], Division of Hematology/Oncology, Department of Medicine [P. M-B., J. M. R.], Department of Pathology [W. G.], and Oral Cancer Research Center and Comprehensive Cancer Center [W. G., T. R. B., L. T. C., J. M. R.], University of Alabama at Birmingham, Birmingham, Alabama 35294-3300
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Abstract |
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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One limitation to the isolation of oncogenes has been the paucity of in vitroassays for functional expression cloning. Whereas most studies have used NIH3T3 or other mesenchymal cells as host for analysis of oncogenes relevant to carcinoma, the potential use of a host cell with epithelial characteristics has been discussed (2) . In addition, several known oncogenes exhibit cell-type specificity. GLI, BCR-ABL, NOTCH1/TAN1, and the G protein GIP2 have been found to transform immortalized rat cells (14, 15, 16, 17, 18) , but not NIHT3 cells, demonstrating the potential use of alternate assays for oncogene expression cloning.
A consistent feature of human tumors is inactivation of the G1 phase cell cycle regulatory pathway that includes p105Rb (19, 20, 21, 22) . Loss-of-function mutations affect p105Rb or the cyclin-dependent kinase inhibitors, or gain-of-function mutations occur in cyclin-dependent kinases or associated cyclins. Such alterations are rate-limiting for tumor formation in vivo because inheritance of these defects predisposes to retinoblastoma, cutaneous malignant melanoma, and other tumors. During viral infection of normal cells, disruption of the same pathway is critical for successful induction of the cellular DNA replicative machinery to support viral replication. Therefore, viruses express proteins, such as adenovirus E1A, that affect cell cycle progression through direct interaction with cell cycle regulators including p105Rb, p27Kip1, and others (23, 24, 25, 26) .
We previously developed and used RK3E cells, immortalized by E1A, to demonstrate the transforming activity of GLI (17) . We now show that these cells exhibit multiple features of epithelia and detect known and novel transforming activities in tumor cell lines. The epithelial features of the cells and/or the mechanism of immortalization may explain the surprising sensitivity and specificity of the assay compared with previous expression cloning approaches (27) . Three of the four genes known to transform RK3E cells are activated by genetic alterations in carcinomas, and, of these genes, only RAS exhibits transforming activity in the commonly used host NIH3T3. We identify GKLF4 (3) as an oncogene that is expressed in the differentiating compartment of epithelium and misexpressed in dysplastic epithelium. We also suggest that GKLF may regulate the rate of differentiation and maturation and the overall cellular transit time through epithelium. The functional similarities shared with other oncogenes, including GLI or c-MYC, identify GKLF as an attractive candidate gene relevant to tumor pathogenesis.
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Results |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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. Northern blot analysis showed that RK3E cells, but not REF52 fibroblasts, expressed desmoplakin, a major component of desmosomes and an epithelial marker (Fig. 1B)
. By immunocytochemical staining, the mesenchymal marker vimentin was low or undetectable in RK3E cells, but was strongly positive in REF52 cells (Fig. 1C)
. Neither line reacted strongly with anticytokeratin or antidesmin antibodies. These results are consistent with the observation that E1A induces multiple epithelial characteristics without inducing cytokeratin expression (28)
.
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cDNA Library Construction.
To identify transforming genes, we used mRNA from human squamous cell carcinoma- or breast tumor-derived cell lines. These tumor types do not exhibit frequent alteration of RAS or GLI. After pooling mRNAs for each tumor type, oligo dT-primed cDNA libraries were constructed in bacteriophage 
(Table 1)
. The libraries were high-titer (assessed before amplification on agar plates) with a mean insert size of 1.6–1.7 kb. The amplified breast cDNA library was further assessed by plaque screening for the transcription factor hBRF, using a probe derived from the 5' end of the protein coding region (bases 315–655, accession U75276). Each of the seven clones identified were derived from independent reverse transcripts, as determined by end sequencing, confirming that complexity of the library was maintained during amplification. The inserts ranged in size from 2.1–3.4 kb and contained the entire 3' UTR and much or all of the protein coding region intact. Three of the seven clones extended through the predicted initiator methionine codon, whereas four others were truncated further downstream. These results suggested that the library is relatively free of COOH-terminally truncated clones and contains full-length cDNAs even for relatively long mRNAs. The overall abundance of hBRF mRNA has not been determined.
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. Eleven of these PCR products were identified as human c-MYC by end-sequencing and restriction enzyme analysis. The c-MYC cDNA in Lane 15 included the coding region and 193 bases of 5' UTR sequence (accession V00568). As determined by sequencing or restriction mapping, the other c-MYC cDNAs extended further 5' (Lanes 1, 3, 5–7, 9–7, 13, and 14), such that all of the clones contained the entire protein-coding region.
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, Lanes 8 and 12) contained cDNAs coding for GKLF. Mouse and human GKLF cDNAs were previously isolated by hybridization with zinc finger consensus probes (30, 31, 32)
, but were not implicated as oncogenes or found to be induced during neoplastic progression. After cloning into plasmid, the sequences of these two cDNAs, termed SQC7 and SQC11, were obtained in total. As determined by comparison with multiple expressed sequence tags and two full-length coding sequence files in the database (accessions U70663 and AFO22184), each contained the predicted GKLF protein coding region bounded by 5' and 3' UTRs. An ATG in good context for translation initiation was located at base 330, with the predicted terminator codon at base 1740. Both isolates were artificially truncated at the XhoI site in the 5' UTR during library preparation. Because the transcripts had been processed using distinct AAUAAA polyadenylation signals, the cDNAs were slightly different in length and derived from independent mRNA molecules (Fig. 2A)
. Sequencing revealed these two GKLF isolates to be identical within the residual 5' UTR and throughout the coding region. A single bp difference in the 3' UTR represents a PCR-induced error or a rare variant, as determined by comparison with ESTs. Comparison to a placenta-derived sequence (accession U70663) revealed three single bp differences in the coding region. These differences were resolved by alignment with other sequences in the database (accessions AF022184 and AA382289) from normal tissues, indicating that the GKLF molecules obtained by expression cloning are predicted to encode the wild-type protein.
Reconstitution of Transforming Activity for c-MYC and GKLF.
To demonstrate transforming activity, three independent PCR products each for the c-MYC and GKLF cDNAs were cloned into the retroviral expression vector pCTV3K (27)
, packaged into virions, and tested for transformation of RK3E cells in vitro (Fig. 2, B and C
; Table 2
). One of the c-MYC clones (pCTV3K-SQC1) possessed greatly reduced transforming activity in multiple experiments despite similar viral titers, as determined by induction of hygromycin resistance, suggesting that an error may have been introduced during PCR. Each of the other virus supernatants carrying GKLF and c-MYC transgenes induced >1000 foci/dish compared with no foci for virus controls.
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. The c-MYC-transduced cells exhibited loss of contact inhibition and dense growth in 89% of colonies. The GKLF-transduced cells exhibited a transformed morphology in 44% of colonies. In comparison, a previous study showed that 70% and 40% of NIH3T3 colonies transduced by viruses carrying RAS and RAF exhibited a transformed morphology (27)
. We, likewise, tested virus supernatants for transformation of NIH3T3 cells. Neither c-MYC nor GKLF induced morphological transformation of NIH3T3 colonies, as previously described for GLI and others (data not shown; Ref. 17
). These results identify the RK3E assay as not only highly specific, but also sensitive to the activity of a select group of oncogenes.
In lieu of sequencing the c-MYC alleles, we confirmed that wild-type c-MYC can transform RK3E cells. A human wild-type expression vector (pSR
MSV c-MYC tk-neo) induced foci using direct plasmid transfection of RK3E cells in multiple experiments. Foci were observed at a similar frequency using known wild-type or new c-MYC isolates when analyzed in parallel (data not shown). In addition, retrovirus encoding the estrogen receptor-c-MYC (wild-type) fusion protein induced morphological transformation of RK3E cells in the presence or absence of 4-hydroxy-tamoxifen (33)
. No effect was observed for controls (empty vector or a control containing a deletion in c-MYC residues 106–143).
Northern blot analysis of transformed RK3E cell lines demonstrated expression of the c-MYC and GKLF vector-derived transcripts (Fig. 3A)
. No endogenous transcripts were detected at the stringency used in this experiment. Compared with RK3E cells at subconfluence (Lane 1) or confluence (Lane 2), no consistent increase of E1A transcripts was detected in cells transformed by RAS, GLI, c-MYC, or GKLF, suggesting that these genes act upon cellular targets to induce transformation.
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Cell lines derived from foci induced by c-MYC or GKLF were further tested for tumorigenicity in athymic mice by s.c. inoculation at four sites for each line (Table 3
; Ref. 17
). Tumors were >1 cm in diameter and were scored at 2–4 weeks after inoculation. Cells transformed by c-MYC induced tumors in 75% or 100% of sites injected (two lines tested). Three lines transformed by GKLF each induced tumors in 50–75% of sites injected. No tumors resulted from injection of RK3E cells, whereas a GLI-transformed cell line induced tumors in each of the four sites injected. In all, GKLF cell lines induced tumors in 8 of 12 injection sites, compared with 7 of 8 injection sites for c-MYC and 4 of 4 injection sites for GLI. GKLF-induced tumors also grew more slowly in vivo, reaching 1 cm in diameter by 3.4 weeks, on average, compared with 2.6 weeks for c-MYC and 3 weeks for GLI. The moderately increased latency and decreased efficiency of tumor formation for GKLF cell lines may be attributable to the intrinsic rate of proliferation for these cells (Table 3)
. Although c-MYC, GLI, and GKLF cell lines all exhibited prolonged doubling times in vitro compared with RK3E cells, GKLF cells divided more slowly than the other transformed cell lines.
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. GKLF expression in breast or squamous cell carcinoma cell lines was variable, with increased expression in the breast tumor line ZR75–1 and the squamous cell lines SCC15 and SCC25 (Fig. 3B)
. In human squamous cell carcinomas microdissected to enrich for tumor cells, GKLF expression was detected in each of 10 primary or metastatic tumors analyzed, with expression levels comparable with that for the cell line SCC25 (Fig. 3C)
. The results suggest that GKLF represents a potent transforming activity that is consistently expressed in tumors as well as in tumor-derived cell lines. Because GKLF was isolated from cell lines that express the gene at a level found in tumors in vivo, the results suggest that GKLF may represent a major transforming activity in tumors, as well as in cell lines.
Gene Copy Number of c-MYC and GKLF.
c-MYC was previously shown to be activated by gene amplification in 
10% of oral squamous cancers and may be activated in these or other tumors by genetic alteration of WNT-APC-ß-catenin pathway components (6
, 34, 35, 36, 37)
. To determine whether expression of GKLF in cell lines and tumors is, likewise, associated with gene amplification, we performed Southern blot analysis (Fig. 4, A and B)
. Filters were sequentially hybridized to GKLF, c-MYC, and ß-tubulin. Increased copies of c-MYC were identified in two cell lines used for library construction, FaDu and MCF7. Increased hybridization to c-MYC was, likewise, observed for 1 of 11 oral squamous cell carcinomas (Fig. 4A
, Lane 10) and for one of nine breast carcinomas (Fig. 4B
, Lane 8). These results are consistent with the published frequencies of c-MYC amplification for these tumor types (34
, 35
, 38)
. No copy number gains of GKLF were observed, indicating that other mechanisms may contribute to expression of GKLF in tumors. The same may be true for c-MYC because gene amplification in FaDu cells was associated with reduced expression compared with other oral cancer cell lines (Fig. 3B)
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; Table 4
). No specific GKLF expression was detected in the basal or parabasal cells or in the underlying dermis. In contrast, a sense control probe produced grains at a much-reduced frequency in a uniform fashion across the epithelium. GAPDH expression served as a positive control and was detected diffusely throughout the entire epithelium (data not shown). The observed pattern of GKLF expression is identical to the pattern in normal mouse skin (32)
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; Table 4
, Cases 1, 2, 4, 9, 11, 12, and 15–17). In contrast to results obtained in normal-appearing epithelium, there was no reduction of expression in the basal and parabasal layers compared with superficial layers. For tissue sections that contained both uninvolved epithelium and adjacent dysplastic epithelium, the overall level of GKLF expression in dysplastic epithelium was prominently elevated compared with the GKLF-positive cell layers in uninvolved epithelium (Fig. 5, B, E, and H
; Table 4
, Cases 1, 2, 4, 11, 12, and 16). These results suggest that GKLF expression is qualitatively and quantitatively altered in dysplasia, that exclusion of GKLF from the basal and parabasal cell layers is lost early during neoplastic progression, and that GKLF exhibits properties of an oncogene not only in vitro, but also in vivo.
As shown by Northern blot analysis, GKLF transcripts are consistently present in tumor-derived mRNA (Fig. 3C
; Table 4
). To determine whether GKLF is expressed in tumor cells, we examined laryngeal squamous cell carcinomas by mRNA ISH. Expression was detected in each primary (13 cases) or metastatic (5 cases) tumor examined (Fig. 5, J–O
; Table 4
), with all or nearly all tumor cells associated with silver grains. The level of expression was somewhat heterogeneous, with higher levels found in the periphery and in nodules of tumor containing centrally necrotic cells or keratin pearls. As for dysplastic epithelium, expression in tumor cells was consistently elevated compared with uninvolved epithelium in the same sections (Fig. 5, H and K
; Table 4
, Cases 1, 2, 11, 12, and 16). However, expression in tumor cells was not higher than in dysplastic epithelium (Cases 1, 9, 11, 12, and 15–17). For several cases, expression in the most dysplastic epithelium was higher than in adjacent GKLF-positive tumor, suggesting that GKLF expression is specifically activated during the transition from normal epithelium to dysplasia, before invasion or metastasis.
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and MethodsReferences |
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Whitehead et al. (27)
developed the retroviral vectors that we used in this study for transduction of NIH3T3 cells, in which they isolated 19 different cDNAs encoding 14 different proteins. Known oncogenes were isolated, including raf-1, lck, and ect2. Other known genes included phospholipase C-
2, ß-catenin, and the thrombin receptor. In addition to the known genes, seven novel cDNAs were isolated, including several members of the CDC24 family of guanine nucleotide exchange factors. Only the thrombin receptor was isolated more than once, and many of the 14 different genes identified were truncated within the protein coding region. The diversity of cDNAs isolated in the NIH3T3 assay is in contrast to results obtained in the current study. The specificity of the RK3E assay may be attributable to the "tumor suppressor" activity of the E1A oncogene (28
, 45)
. Although E1A antagonizes p105Rb and immortalizes primary cells, it also induces epithelial differentiation in diverse tumor types, including sarcoma, and suppresses the malignant behavior of tumor cells in vivo.
GKLF was previously isolated by hybridization to zinc finger probes (30, 31, 32) . The human gene is located at chromosome 9q31 and is closely linked to the autosomal dominant syndrome of multiple self-healing squamous epitheliomata (31 , 32 , 46 , 47) . Affected individuals develop recurrent invasive, but well-differentiated, tumors morphologically similar to squamous carcinoma that spontaneously regress. Although GKLF has been proposed as a candidate tumor suppressor gene relevant to multiple self-healing squamous epitheliomata (32) , our results suggest that activating mutations could account for the syndrome.
GKLF encodes a nuclear protein that functions as a transcription factor when bound to a minimal essential binding site of 5'-G/AG/AGGC/TGC/T-3' (48) . The 470 residue polypeptide exhibits modular domains that mediate nuclear localization, DNA binding, and transcriptional activation or repression (31 , 32 , 49 , 50) . In mice, GKLF expression is found predominately in barrier epithelia, including mucosa of the mouth, pharynx, lung, esophagus, and small and large intestine (30 , 32) . A role for GKLF in differentiation or growth arrest was suggested by the onset of expression at the time of epithelial differentiation (approximately embryonic day 13; Refs. 32 and 51 ) and by similarity within the zinc finger domain to family members erythroid Krüppel-like factor and lung Krüppel-like factor that were previously associated with growth-arrest or differentiation-specific gene expression (52 , 53) . Similarity to these other genes is limited to the DNA-binding zinc finger region.
Our results show that GKLF can induce proliferation when overexpressed in vitro. Analysis of expression in dysplastic cells and tumor cells in vivo provides independent evidence that GKLF exhibits properties expected of an oncogene. Genetic progression of carcinoma seems to involve genes and pathways important for homeostasis of normal epithelium (6 , 7 , 9 , 54) . For example, the zinc finger protein GLI is expressed in normal hair shaft keratinocytes, whereas c-MYC is expressed in normal epithelium of the colonic mucosa. In tumors derived from these tissues, GLI and c-MYC are more frequently activated by recessive genetic changes in upstream components of their respective biochemical pathways than by gain-of-function alterations such as gene amplification. Up-regulation of GKLF expression in dysplastic epithelium and tumor cells in vivo is particularly interesting as expression seems not to be increased by proliferation in vitro. Expression of the endogenous GKLF mRNA in RK3E cells was similar in cycling versus contact-inhibited cells (data not shown). In contrast, GKLF is significantly induced in NIH3T3 cells during growth arrest (30) . These different results suggest that cell type-specific mechanisms can regulate GKLF expression, and that GKLF may play different roles in epithelial versus mesenchymal cells.
Squamous epithelium is divided into compartments (55 , 56) . In the basal cell layer, proliferative reserve or stem cells possess long-term or unlimited self-renewal capacity, whereas the parabasal transit amplifying cells undergo several rounds of mitosis and then withdraw from the cell cycle to differentiate into spinous cells that form the mid strata of the epithelium. These cells then undergo terminal differentiation and programmed cell death at the surface. Proliferation and differentiation are normally balanced such that overall cell number remains constant. In contrast to GLI and c-MYC, GKLF expression in skin seems limited to the differentiating compartment (32) . A simple model is that GKLF normally regulates the rate of maturation and shedding and the overall transit time for individual cells. The thickness of epithelium, which varies greatly in development and in different adult tissues, may be regulated not only by alterations in the rate of cell division in the basal layer, but also in response to GKLF or similarly acting molecules in the suprabasal layers. This model is consistent with the relatively late induction of GKLF during mouse development, and is testable by modulating expression of GKLF in transgenic animals or using raft epithelial cultures in vitro. Activation of GKLF in the basal layer of dysplastic epithelium suggests that dysplasia and progression to invasion and metastasis could result from loss of normal compartment-specific patterns of gene expression.
In summary, GKLF, c-MYC, and GLI are potent oncogenes in epithelioid RK3E cells in vitro, are analogous with respect to their expression in normal epithelium, and have potentially complex roles in the regulation of epithelial cell proliferation, differentiation, or apoptosis (6 , 7 , 9 , 44 , 56, 57, 58) . How GKLF contributes to these processes will require a better understanding of its function and of the pathways that regulate GKLF activity in epithelia.
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Materials and Methods |
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Construction of cDNA Libraries.
Two cDNA libraries were constructed using the ZAP-Express cDNA synthesis kit (Stratagene, La Jolla, CA). A library was prepared from human squamous cell carcinoma cells derived from tumors of the oro-pharynx. Equal quantities of total mRNA from cell lines SCC15, SCC25, and FaDu (American Type Culture Collection, Manassas, VA) were pooled. Similarly, equal quantities of mRNA from the breast cancer cell lines MCF-7, ZR75–1, MDAMB-453, and T47D (American Type Culture Collection) were pooled. For each pool, poly(A)+ mRNA was selected by two cycles of oligo-dT cellulose affinity chromatography, and 5 µg were reverse transcribed using an oligo-dT linker primer and MMLV reverse transcriptase. Double-stranded cDNA was synthesized using Escherichia coli RNase H and DNA polymerase I. cDNA was ligated to ZAP EXPRESS bacteriophage arms and packaged into virions. The titer and the frequency of nonrecombinants were determined before amplification of the library on bacterial plates (Table 1)
. The frequency of nonrecombinant clones was estimated to be <2% by complementation of ß-gal activity (blue/white assay). Phage were converted to pBKCMV plasmids by autoexcision in bacteria. Insert sizes in randomly selected clones were determined at this step by gel electrophoresis of plasmid DNA digested with SalI and NotI (Table 1)
. The pBKCMV plasmid libraries were amplified in soft agar at 4 x 104 colony forming units/ml (27)
. After incubation at 37°C for 15 h, bacterial cells within the agar bed were isolated by centrifugation, amplified for 3–4 doublings in culture, and plasmid DNA was purified using a Qiagen column (Qiagen, Inc., Chatsworth, CA).
To generate libraries in a retroviral expression vector, cDNA inserts were excised from 10 µg of plasmid using SalI and XhoI. After treatment with Klenow and dNTPs and extraction with phenol, the DNA was ligated to 5' phosphorylated BstXI adapters (5'-TCAGTTACTCAGG-3' and 5'-CCTGAGTAACTGACACA-3'), as described (27) . After treatment with NotI, excess adapters were removed by gel filtration, and the residual vector was converted to a 9.0-kb dimer using the NotI site and T4 DNA ligase. The cDNA was size-fractionated by electrophoresis in Sea Plaque agarose (FMC BioProducts, Rockland, ME) and fragments 0.6–8.5 kb were isolated and ligated to the BstXI- and alkaline phosphatase-treated MMLV retroviral vector pCTV1B (27) . E. coli MC1061/p3 were transformed by electroporation and selected in soft agar as above.
Retroviral Transduction.
The libraries were analyzed in two transfection experiments performed on consecutive days. For each library, ten 10-cm dishes of BOSC23 ecotropic packaging cells at 80%–90% confluence were transfected using 30 µg of plasmid DNA/dish (29)
. The transfection efficiency for these cells was 60%, as determined using a ß-gal control plasmid. Viruses were collected in a volume of 9.0 ml/dish at 36–72 h after transfection, filtered, and the 9.0 ml was expressed into a 10 cm dish containing RK3E cells at 30% confluence. Polybrene was added to a final concentration of 10 µg/ml. After 15 h, and every 3 days thereafter, the cells were fed with growth media (17)
. A total of 20 RK3E dishes were transduced for each library. A ß-gal retroviral plasmid transduced at least 20–30% of RK3E cells in control dishes. For colony assays, hygromycin was used at 100 µg/ml. Cell proliferation rates for transformed cell lines was measured by plating 2 x 105 cells in duplicate and counting cells 96 h later using a hemacytometer (Table 3)
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PCR Recovery of Proviral Inserts.
PCR reactions used 200 ng of cell line genomic DNA, 20 mM Tris-HCl (pH 8.8), 87 mM potassium acetate, 1.0 mM MgCl2, 8% glycerol, 2% DMSO, 0.2 mM of each dNTP, 32 pmol of each primer (5'-CCTCACTCCTTCTCTAGCTC-3'; 5'-AACAAATTGGACTAATCGATACG-3'; Ref. 27)
, 5 units of Taq polymerase (Life Technologies, Inc., Gaithersburg, MD), and 0.3 units of Pfu polymerase (Stratagene, La Jolla, CA) in a volume of 0.05 ml. Cycling profiles were: 95°C for 1 min; then 95°C for 10 s, 59°C for 40 s, 68°C for 8 min (35 cycles).
RNA Extraction and Northern Blot Analysis.
Tumor samples were obtained through the Tissue Procurement Facility of the University of Alabama at Birmingham Comprehensive Cancer Center and the Southern Division of the Cooperative Human Tissue Network. Microdissection was used to isolate tissue composed of >70% tumor cells. Total RNA was isolated as described (59)
, then denatured and separated on a 1.5% formaldehyde agarose gel and transferred to nitrocellulose (Schleicher and Schuell, Keene, NH). Prehybridization was at 42°C for 3 h in 50% formamide, 4 x SSC [SSC is 150 mM NaCl, 15 mM sodium citrate (pH 7.5)], 0.1 M sodium phosphate (pH 6.8), 0.1% sodium PPi, 0.1% SDS, 5 x Denhardt’s, and 25 µg/ml denatured salmon sperm DNA. Hybridization was at 42°C for 16–20 h. The hybridization mixture contained 45% formamide, 4 x SSC, 0.1 M sodium phosphate (pH 6.8), 0.075% sodium PPi, 0.1% SDS, 10% dextran sulfate, and 100 µg/ml denatured salmon sperm DNA. After hybridization, the filter was washed twice in 2 x SSC, 0.1% SDS for 20 min at room temperature, then washed in 0.3 x SSC, 0.3% SDS for 30 min at 59°C (for detection of rat transcripts) or 65°C. For stripping of hybridized probes, the filter was placed in a solution of 2 x SSC, 25 mM Tris-HCl (pH 7.5), 0.1% SDS at initial temperature of 95°C, and shaken for 10 min at room temperature.
ISH.
ISHs were conducted as described (60)
, using sense and antisense 35S-labeled riboprobes generated from a 301-bp EcoRI fragment derived from the GKLF 3' UTR positioned 40 bases from the stop codon. A GAPDH antisense probe corresponding to bases 366–680 (accession M33197) was synthesized using a commercially available template (Ambion, Inc., Austin, TX). All results were obtained in duplicate. High stringency washes were in 0.1 x SSC and 0.1% (v/v) 2-mercaptoethanol at 58°C for GKLF or 68°C for GAPDH. Slides were coated with emulsion and exposed for 14 days.
Nucleotide Sequencing.
Automated sequence analysis was performed for the two independent GKLF isolates using vector-derived primers and sense or antisense primers spaced at 400-bp intervals within the inserts. The complete sequence was obtained for both clones, with one of the clones analyzed for both strands. Primer sequences are available upon request. GKLF sequence was submitted to GenBank (accession AF105036).
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Acknowledgments |
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Footnotes |
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1 Supported by NIH Grant R29 CA65686 (to J. M. R.) and the University of Alabama at Birmingham Oral Cancer Research Center (DE 11910). K. W. F. is a Medical Scientist Training Program trainee (NIGMS T32GM08361-0607). Core Facilities at The University of Alabama at Birmingham are partially supported by the Comprehensive Cancer Center (5P50 CA13148). 
2 Present address: Salk Institute for Biological Studies, La Jolla, CA 92037.
3 To whom requests for reprints should be addressed, at Department of Medicine, Room 570 WTI, University of Alabama at Birmingham, Birmingham, AL 35294-3300. Phone: (205) 975-0556; Fax: (205) 934-9573; E-mail: mruppert{at}uab.edu
4 The abbreviations used are: GKLF, gut-enriched Krüppel-like factor; ß-gal, ß-galactosidase; UTR, untranslated region; MMLV, Moloney murine leukemia virus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ISH, in situ hybridization.
Received for publication 12/21/98. Revision received 4/ 2/99. Accepted for publication 4/15/99.
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subunit of GIP2 induces neoplastic transformation of RAT1 cells. Proc. Natl. Acad. Sci. USA, 88: 7031-7035, 1991.This article has been cited by other articles:
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 S. Liu, H. Zhang, L. Zhu, L. Zhao, and Y. Dong Kruppel-Like Factor 4 Is a Novel Mediator of Selenium in Growth Inhibition Mol. Cancer Res., February 1, 2008; 6(2): 306 - 313. [Abstract] [Full Text] [PDF]
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P. M. Evans, W. Zhang, X. Chen, J. Yang, K. K. Bhakat, and C. Liu Kruppel-like Factor 4 Is Acetylated by p300 and Regulates Gene Transcription via Modulation of Histone Acetylation J. Biol. Chem., November 23, 2007; 282(47): 33994 - 34002. [Abstract] [Full Text] [PDF]
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L. Zhang, A. Wali, C. V. Ramana, and A. K. Rishi Cell Growth Inhibition by Okadaic Acid Involves Gut-Enriched Kruppel-like Factor Mediated Enhanced Expression of c-Myc Cancer Res., November 1, 2007; 67(21): 10198 - 10206. [Abstract] [Full Text] [PDF]
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 W. Ai, H. Zheng, X. Yang, Y. Liu, and T. C. Wang Tip60 functions as a potential corepressor of KLF4 in regulation of HDC promoter activity Nucleic Acids Res., September 25, 2007; 35(18): 6137 - 6149. [Abstract] [Full Text] [PDF]
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R. Rajhans, S. Nair, A. H. Holden, R. Kumar, R. R. Tekmal, and R. K. Vadlamudi Oncogenic Potential of the Nuclear Receptor Coregulator Proline-, Glutamic Acid-, Leucine-Rich Protein 1/Modulator of the Nongenomic Actions of the Estrogen Receptor Cancer Res., June 1, 2007; 67(11): 5505 - 5512. [Abstract] [Full Text] [PDF]
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X. Wei, H. Xu, and D. Kufe Human Mucin 1 Oncoprotein Represses Transcription of the p53 Tumor Suppressor Gene Cancer Res., February 15, 2007; 67(4): 1853 - 1858. [Abstract] [Full Text] [PDF]
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 V. H. Cowling, S. Chandriani, M. L. Whitfield, and M. D. Cole A Conserved Myc Protein Domain, MBIV, Regulates DNA Binding, Apoptosis, Transformation, and G2 Arrest Mol. Cell. Biol., June 1, 2006; 26(11): 4226 - 4239. [Abstract] [Full Text] [PDF]
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N. D. Hendrix, R. Wu, R. Kuick, D. R. Schwartz, E. R. Fearon, and K. R. Cho Fibroblast Growth Factor 9 Has Oncogenic Activity and Is a Downstream Target of Wnt Signaling in Ovarian Endometrioid Adenocarcinomas Cancer Res., February 1, 2006; 66(3): 1354 - 1362. [Abstract] [Full Text] [PDF]
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 D. Wei, M. Kanai, S. Huang, and K. Xie Emerging role of KLF4 in human gastrointestinal cancer Carcinogenesis, January 1, 2006; 27(1): 23 - 31. [Abstract] [Full Text] [PDF]
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Z. Y. Chen, X. Wang, Y. Zhou, G. Offner, and C.-C. Tseng Destabilization of Kruppel-Like Factor 4 Protein in Response to Serum Stimulation Involves the Ubiquitin-Proteasome Pathway Cancer Res., November 15, 2005; 65(22): 10394 - 10400. [Abstract] [Full Text] [PDF]
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 I.-M. Shih, J. J.-C. Sheu, A. Santillan, K. Nakayama, M. J. Yen, R. E. Bristow, R. Vang, G. Parmigiani, R. J. Kurman, C. G. Trope, et al. Amplification of a chromatin remodeling gene, Rsf-1/HBXAP, in ovarian carcinoma PNAS, September 27, 2005; 102(39): 14004 - 14009. [Abstract] [Full Text] [PDF]
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 N. Maeda, W. Fu, A. Ortin, M. de las Heras, and H. Fan Roles of the Ras-MEK-Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase-Akt-mTOR Pathways in Jaagsiekte Sheep Retrovirus-Induced Transformation of Rodent Fibroblast and Epithelial Cell Lines J. Virol., April 1, 2005; 79(7): 4440 - 4450. [Abstract] [Full Text] [PDF]
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D. Wei, W. Gong, M. Kanai, C. Schlunk, L. Wang, J. C. Yao, T.-T. Wu, S. Huang, and K. Xie Drastic Down-regulation of Kruppel-Like Factor 4 Expression Is Critical in Human Gastric Cancer Development and Progression Cancer Res., April 1, 2005; 65(7): 2746 - 2754. [Abstract] [Full Text] [PDF]
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H. Wang, L. Yang, Md. S. Jamaluddin, and D. D. Boyd The Kruppel-like KLF4 Transcription Factor, a Novel Regulator of Urokinase Receptor Expression, Drives Synthesis of This Binding Site in Colonic Crypt Luminal Surface Epithelial Cells J. Biol. Chem., May 21, 2004; 279(21): 22674 - 22683. [Abstract] [Full Text] [PDF]
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A. Y. Pandya, L. I. Talley, A. R. Frost, T. J. Fitzgerald, V. Trivedi, M. Chakravarthy, D. C. Chhieng, W. E. Grizzle, J. A. Engler, H. Krontiras, et al. Nuclear Localization of KLF4 Is Associated with an Aggressive Phenotype in Early-Stage Breast Cancer Clin. Cancer Res., April 15, 2004; 10(8): 2709 - 2719. [Abstract] [Full Text] [PDF]
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I. D. Louro, E. C. Bailey, X. Li, L. S. South, P. R. McKie-Bell, B. K. Yoder, C. C. Huang, M. R. Johnson, A. E. Hill, R. L. Johnson, et al. Comparative Gene Expression Profile Analysis of GLI and c-MYC in an Epithelial Model of Malignant Transformation Cancer Res., October 15, 2002; 62(20): 5867 - 5873. [Abstract] [Full Text] [PDF]
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K. W. Foster, A. R. Frost, P. McKie-Bell, C.-Y. Lin, J. A. Engler, W. E. Grizzle, and J. M. Ruppert Increase of GKLF Messenger RNA and Protein Expression during Progression of Breast Cancer Cancer Res., November 1, 2000; 60(22): 6488 - 6495. [Abstract] [Full Text]
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J.-L. Shie, Z. Y. Chen, M. J. O'Brien, R. G. Pestell, M.-E. Lee, and C.-C. Tseng Role of gut-enriched Kruppel-like factor in colonic cell growth and differentiation Am J Physiol Gastrointest Liver Physiol, October 1, 2000; 279(4): G806 - G814. [Abstract] [Full Text] [PDF]
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 I. D. Louro, P. McKie-Bell, H. Gosnell, B. C. Brindley, R. P. Bucy, and J. M. Ruppert The Zinc Finger Protein GLI Induces Cellular Sensitivity to the mTOR Inhibitor Rapamycin Cell Growth Differ., July 1, 1999; 10(7): 503 - 516. [Abstract] [Full Text]
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X. Chen, D. C. Johns, D. E. Geiman, E. Marban, D. T. Dang, G. Hamlin, R. Sun, and V. W. Yang Kruppel-like Factor 4 (Gut-enriched Kruppel-like Factor) Inhibits Cell Proliferation by Blocking G1/S Progression of the Cell Cycle J. Biol. Chem., August 3, 2001; 276(32): 30423 - 30428. [Abstract] [Full Text] [PDF]
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