Elevated expression of the nuclear export protein, Crm1 (exportin 1), associates with human oesophageal squamous cell carcinoma.

The nuclear export receptor, Crm1 (exportin 1), is involved in the nuclear translocation of proteins and certain RNAs from the nucleus to the cytoplasm and is thus crucial for the correct localisation of cellular components. Crm1 has recently been reported to be highly expressed in certain types of cancers, yet its expression in oesophageal cancer has not been investigated to date. We investigated the expression of Crm1 in normal and tumour tissues derived from 56 patients with human oesophageal squamous cell carcinoma and its functional significance in oesophageal cancer cell line models. Immunohistochemistry revealed that Crm1 expression was significantly elevated in oesophageal tumour tissues compared to normal tissues and its localisation shifted from predominantly nuclear to nuclear and cytoplasmic. Real­time RT­PCR revealed that Crm1 expression was elevated at the mRNA level. To determine the functional significance of elevated Crm1 expression in oesophageal cancer, its expression was inhibited using siRNA, and a significant decrease in cell proliferation was observed associated with G1 cell cycle arrest and the induction of apoptosis. Similarly, leptomycin B (LMB) treatment resulted in the effective killing of oesophageal cancer cells at nanomolar concentrations. Normal oesophageal epithelial cells, however, were much less sensitive to Crm1 inhibition with siRNA and LMB. Together, this study reveals that Crm1 expression is increased in oesophageal cancer and is required for the proliferation and survival of oesophageal cancer cells.

Cemented versus uncemented hemiarthroplasty for intracapsular hip fractures: A randomised controlled trial in 400 patients.

We undertook a prospective randomised controlled trial involving 400 patients with a displaced intracapsular fracture of the hip to determine whether there was any difference in outcome between treatment with a cemented Thompson hemiarthroplasty and an uncemented Austin-Moore prosthesis. The surviving patients were followed up for between two and five years by a nurse blinded to the type of prosthesis used. The mean age of the patients was 83 years (61 to 104) and 308 (77%) were women. The degree of residual pain was less in those treated with a cemented prosthesis (p < 0.0001) three months after surgery. Regaining mobility was better in those treated with a cemented implant (p = 0.005) at six months after operation. No statistically significant difference was found between the two groups with regard to mortality, implant-related complications, re-operations or post-operative medical complications. The use of a cemented Thompson hemiarthroplasty resulted in less pain and less deterioration in mobility than an uncemented Austin-Moore prosthesis with no increase in complications.

Ectopic Tbx2 expression results in polyploidy and cisplatin resistance.

T-box factors play critical roles in embryonic development and have been implicated in cell cycle regulation and cancer. For example, Tbx2 can suppress senescence through a mechanism involving the repression of the cyclin-dependent kinase inhibitors, p19(ARF) and p21(WAF1/CIP1/SDII), and the Tbx2 gene is deregulated in melanoma, breast and pancreatic cancers. In this study, several transformed human lung fibroblast cell lines were shown to downregulate Tbx2. To further investigate the role of Tbx2 in oncogenesis we therefore stably reexpressed Tbx2 in one such cell line. Compared to their parental cells, the resulting Tbx2-expressing cells are larger, with binucleate and lobular nuclei containing double the number of chromosomes. Moreover, these cells had an increase in frequency of several features of genomic instability such as chromosome missegregation, chromosomal rearrangements and polyploidy. While grossly abnormal, these cells still divide and give rise to cells that are resistant to the chemotherapeutic drug cisplatin. Furthermore, this is shown to be neither species nor cell type dependent, as ectopically expressing Tbx2 in a murine melanoma cell line also induce mitotic defects and polyploidy. These results have important implications for our understanding of the role of Tbx2 in tumorigenesis because polyploidy frequently precedes aneuploidy, which is associated with high malignancy and poor prognosis.

Expression of p53 and its homolog, p73, in HPV DNA positive oesophageal squamous cell carcinomas.

Several studies have detected human papilloma virus (HPV) DNA in squamous cell carcinoma of the oesophagus (OSCC). In this study, we analysed OSCC specimens from 114 patients for the presence of HPV DNA, and p53 and p73 expression. HPV DNA was detected in 44.7% of cases, with the low risk HPV11 occurring most frequently. p53 and p73 expression was detected in 70% and 61.4% of cases, respectively. There was no correlation between expression of p53, p73 or HPV infection and tumour grade, or between p53 expression and the presence of HPV DNA. There was, however, significant correlation between p73 expression and the presence of HPV DNA (p<0.01) and p53 and p73 co-expression (p<0.001), as well as co-expression of p53 and p73 with HPV status (p<0.05). These data support previous studies suggesting a role for HPV infection in OSCC and also indicate that HPV infection and p53 and p73 overexpression are not mutually exclusive. In addition, the data implicate a role for p73 in OSCC and suggest a complex interaction between p53, p73 and HPV in the aetiology of the disease.

Human papillomavirus associated with oesophageal cancer.

AIM: To study the prevalence and the different types of human papillomavirus (HPV) in patients with oesophageal cancer from a high risk area of South Africa (Transkei). METHODS: DNA samples from 50 paraffin wax embedded tissue sections were analysed by nested polymerase chain reaction (PCR) using the degenerate HPV L1 consensus primer pairs MY09/MY11 and GP5+/GP6+. Positive PCR samples were subjected to DNA sequence analysis. RESULTS: HPV DNA was detected in 23 of the 50 samples. Sequence analysis revealed that most patients (11) harboured DNA to HPV type 11, whereas other types included DNA HPV type 39 (seven patients), type 16 (two patients), and type 52 (one patient). HPV type 39 has not previously been shown to be associated with oesophageal cancer. In contrast to earlier studies that have found HPV type 16 to be more frequently associated with oesophageal cancer, HPV type 11 was the predominant subtype in this study. CONCLUSIONS: The high frequency of occurrence of HPV in oesophageal tumours (23 of 50 patients; 46%) implicates HPV as one of the possible aetiological factors in this disease. The finding that the low risk HPV subtypes predominate indicates that transformation may be effected via the E6 and E7 proteins.

Breast tumour cell-induced down-regulation of type I collagen mRNA in fibroblasts.

This study investigated the modulation of type I collagen gene expression in normal fibroblasts by breast tumour cells. Northern analysis of total RNA extracted from stages I, II and III breast tumour tissue revealed that collagen mRNA levels were elevated in stage I tumours compared to the adjacent normal breast tissues, whereas they were decreased in stages II and III breast tumours. This aberrant collagen gene expression was confirmed by non-radioactive RNA:RNA in situ hybridization analysis of 30 breast carcinomas which localized the production of type I collagen mRNA to the stromal fibroblasts within the vicinity of the tumour cells. In order to determine whether the tumour cells were directly responsible for this altered collagen production by the adjacent fibroblasts, breast tumour cell lines were co-cultured with normal fibroblasts for in vitro assessment of collagen and steady-state collagen RNA levels. Co-culture of tumour cells and normal fibroblasts in the same dish resulted in down-regulation of collagen mRNA and protein. Treatment of the fibroblasts with tumour-cell conditioned medium also resulted in decreased collagen protein levels but the mRNA levels, however, remained unaltered. These results suggested that the tumour cells either secrete a labile 'factor', or express a cell surface protein requiring direct contact with the fibroblasts, resulting in down-regulation of collagen gene expression. Modulation of the ECM is a common characteristic of invading tumour cells and usually involves increased production of collagenases by the tumour cells or stromal fibroblasts. This study showed that tumour cells were also able to modulate collagen mRNA production by stromal fibroblasts, which may facilitate tumour cell invasion and metastasis.

CA repeat polymorphism in the promoter region of the COL1A2 gene.

A polymorphic CA dinucleotide repeat sequence has been identified within the promoter of the human alpha 2(I) procollagen gene, located at 7q21.3-q22.1. Nine alleles have been identified in unrelated individuals and the observed heterozygosity for the polymorphism was 0.66. This marker may be useful in the prenatal diagnosis of inherited connective tissue diseases in which the COL1A2 gene is involved. Furthermore, it may potentially improve the usefulness of the COL1A2 genetic system as an anthropogenetic marker.

Mapping of novel regions of DNA gain and loss by comparative genomic hybridization in esophageal carcinoma in the Black and Colored populations of South Africa.

Esophageal cancer (EC) is the leading cause of cancer death in the Black male population in South Africa. Although several oncogenes and tumor suppressor genes have previously been found altered in this cancer, many novel genes remain to be identified. To identify the chromosomal location of these unknown genes, we have analyzed DNA of 29 South African EC patients by comparative genomic hybridization. Frequent loss occurred at chromosome 1p (52%), 4p (52%), 18q (48%), 19p (52%), 19q (55%), and 22q (41%). The most common gains were detected at 1q (41%), 2q (52%), 3q (72%), 5p (31%), 7p (48%), 7q (45%), 8q (55%), and Xq (69%). High level amplification was detected at 2q24-33, 6p21.1-q14, 7p12-q21, 7q11.2-31, 8q22-24, 8q13-qter, 13q21-34, and at 13q32-34. The present comparative genomic hybridization study opens the way for additional targeted studies on these particular chromosomal regions to identify the specific genes involved in the higher susceptibility to specific subtypes of esophageal carcinoma in different geographical regions. The loss of 8p (28%) and Xp (17%) in tumors of male individuals may provide clues to the basis of the sex-biased frequency of occurrence of EC favoring men.

Effect of rooperol on collagen synthesis and cell growth.

The effect of rooperol on type I collagen synthesis in normal skin and lung fibroblasts and cell growth in normal and transformed fibroblasts was investigated. Low concentrations of rooperol selectively inhibited the growth of transformed cells while stimulating collagen synthesis in normal fibroblasts. Elevated collagen synthesis and deposition could impede tumour cell invasion and metastasis, implying that rooperol may be useful as an antimetastatic agent in the treatment of cancer.

Characterization of two distinct families of transcription factors that bind to the CCAAT box region of the human COL1A2 gene.

Both the mouse and human alpha2(I) procollagen promoters contain an inverted CCAAT box at -80, but only the human promoter contains an additional regulatory element, the collagen modulating element (CME), immediately downstream of the CCAAT box [Collins et al. (1997): Biochem J 322:199-206]. In this study, the transcription factors that bind to the G/CBE and CME within the human promoter were characterized in SVWI-38 and CT-1 nuclear extracts. Two distinct proteins bind to the CME, and both were identified as heat-labile factors that were sensitive to high ionic strengths and required Zn2+ for DNA-binding activity. These proteins had Stokes radii of 4.12 and 3.15 nm, sedimentation coefficients of 3.9 and 3.2 S and native molecular weights of 66 and 41 kDa, respectively. On the basis of biochemical and DNA-binding properties, the CME binding proteins are probably novel factors involved in the regulation of the human alpha2(I) procollagen gene. By contrast, the G/CBE binding proteins were more resistant to heat, ionic strength, and divalent metal ion chelators, demonstrating that the G/CBE and CME binding proteins had distinct DNA-binding properties. The above properties suggest that this factor is a member of the previously characterized family of CCAAT box-binding factors, CBF, NF-Y, CP-1 and alpha-CP1. Taken together, these physicochemical properties of the COL1A2 CCAAT box and CME-binding proteins demonstrated that they were distinct unrelated transcription factors. These results also suggest that there is a distinct difference in the DNA-binding activity between the equivalent region of the mouse and human alpha2(I) procollagen promoters.

Regulation of the human alpha 2(1) procollagen gene by sequences adjacent to the CCAAT box.

The human, rat, mouse and chicken alpha 2(1) procollagen promoters analysed to date all contain an inverted CCAAT box at -80. In this study we have examined the binding of nuclear proteins to the proximal promotor of the human alpha 2(1) procollagen gene, where an inverted CCAAT box is flanked by a downstream GGAGG sequence and its inverted counterpart (CCTCC) on the upstream end. Each of the GGAGG sequences is separated from the inverted CCAAT box by a single pyrimidine nucleotide (5'-CCTCCCATTGGTGGAGGCCCTTTT-3'). Electrophoretic mobility-shift assays (EMSAs) revealed that two distinct DNA-protein complexes formed on this DNA sequence. Methylation interference analysis and in vitro mutagenesis studies revealed that the integrity of the sequence 5'-CCTCCCATTGG-3' (the GGAGG/CCAAT-binding element or G/CBE) was important for the binding of the CCAAT-binding factor (CBF) (complex I). Competition studies showed that complex formation on the human G/CBE could be competed by mouse CBE and nuclear factor-Y (NF-Y) oligonucleotides, suggesting that mouse CBE and human G/CBE-binding proteins belong to the same family of CCAAT box binding proteins. Furthermore, antibodies to mouse CBF specifically supershifted the G/CBE complex (complex I) in EMSAs. The downstream GGAGG and 3'-flanking sequences (5'-GGAGGCCCTTTT-3') or collagen modulating element (CME), however, were important for the formation of a novel DNA protein complex (complex III). The formation of this complex was not competed out by CBE or NF-Y oligonucleotides, nor was DNA-protein complex formation affected by the anti-CBF antibody. Functional analysis of G/CBE and CME elements subjected to mutagenesis, using promoter-chloroamphenicol acetyl transferase constructs in transient transfection assays, showed that both these elements were essential for activity of the human promoter. These experiments identified a novel regulatory element in the human alpha 2(1) procollagen gene which is not present in the rodent gene.

Transcriptional repression of the alpha 1(I) collagen gene by ras is mediated in part by an intronic AP1 site.

We have previously shown that transformation of fibroblasts by ras results in transcriptional inhibition of the alpha 1(I) gene. An alpha 1(I)-hGH chimeric plasmid containing 3.7 kb of 5' flanking and 4.4 kb of alpha 1(I) transcribed sequence was regulated appropriately by ras in a transient transfection assay. In contrast, a similar plasmid containing alpha 1(I) DNA from -220 to +500 was virtually unresponsive to ras. The regions from -3700 to -220 and +500 to +4400 contributed equally to the ras-mediated inhibition of the parental plasmid. Deletion analysis indicated that a short fragment, between +500 and +890 in the first intron of the alpha 1(I) gene, was recognized differently in ras-transformed and wild-type cells. A previously described AP1 site in this fragment stimulated alpha 1(I) transcription in Rat1 fibroblasts but was inactive in ras-transformed cells. Mobility shift assays using nuclear extracts from the two cell types demonstrated differences in binding to the alpha 1(I) AP1 site. We conclude that ras transformation suppresses the function of a cell-specific enhancer in the first intron of the alpha 1(I) collagen gene.

Identification of cardiac autoantigens in human heart cDNA libraries using acute rheumatic fever sera.

Antigenic mimicry or cross-reactivity between Group A streptococcal antigens and cardiac autoantigens may initiate an autoimmune response resulting in cardiovascular damage in acute rheumatic fever. This study describes a molecular biological approach to the identification of such cross-reactive cardiac antigens. Two human heart cDNA libraries were constructed in the expression vector lambda gt11 and screened with patient sera, monoclonal antibodies and rabbit immune sera cross-reactive with streptococcal and cardiac antigens. Using the serum of a patient with a recurrent acute attack of rheumatic fever containing high titres of antibodies cross-reactive with both sets of antigens, we were able to identify three positive clones with insert sizes of 1.0 kb, 1.4 kb and 0.9 kb in these libraries. Acute rheumatic fever (ARF) sera reacted more strongly with these autoantigen clones than did normal sera. Autoantibodies eluted from the purified plaques of all three clones displayed different patterns of cross-reactivity against immunoblots of streptococcal M5, M6, M19 and M24 protein extracts. The cDNA inserts were sequenced and compared with known sequences in the EMBL and Genbank databases. One clone was 98% homologous with human cytokeratin 8 and showed homologies of 40 to 50% with human cardiac heavy chain myosin, tropomyosin and streptococcal M5 protein--all members of the alpha-helical coiled-coil family of proteins. Another clone was completely homologous to the G-protein alpha-subunit of adenyl cyclase, whilst the sequence of the third clone was not found in any of the data banks.

Selective hypermethylation of transcribed nucleosomal DNA by sodium butyrate.

Previous studies have shown that treatment of cultured fibroblasts with millimolar concentrations of sodium butyrate results in increased methylation of cytosine residues in DNA. In this study, active nucleosomes were fractionated from the inactive ones by organomercurial agarose column chromatography. DNA in each fraction was hydrolyzed to its constituent bases and subjected to HPLC analysis in order to determine the 5-methylcytosine content. In control cells, the active nucleosomal DNA was hypomethylated (0.97 +/- 0.27% 5-methyleytosine) when compared with the inactive DNA fraction (1.61 +/- 0.15%). This result was not unexpected since DNA hypermethylation is generally associated with gene inactivation. Treatment of cells with sodium butyrate, however, resulted in increased methylation of the active nucleosomal DNA such that it was comparable to that of the inactive fraction of control cells (1.73 +/- 0.02% 5-methylcytosine). A much smaller increase in 5-methylcytosine content was detected in the inactive DNA fraction of sodium butyrate-treated cells (from 1.61 to 1.89%). Removal of the sodium butyrate followed by a chase in butyrate-free medium for up to 120 h failed to reverse the butyrate-induced hypermethylation. Reversal was achieved only after continuous culture in butyrate-free medium for 10 days.

Protected regions in the chicken alpha 2(1) procollagen promoter in differentiated tissues.

The higher ordered structure of the chicken alpha 2(1) procollagen gene was analyzed in chromatin isolated from expressing (lung) and nonexpressing (reticulocyte and erythrocyte) tissues. Digestion of DNA with methylation sensitive restriction endonucleases revealed that this gene was methylated in all tissues examined and that no differences existed in the promoter methylation patterns between expressing and nonexpressing tissues. DNAse 1 hypersensitive sites were located between 100-300 bp upstream from the transcription initiation site and within the first intron. These sites were also hypersensitive to the single-strand specific S1 nuclease, implying that this region of the gene in the chromatin is either in an unfolded single-stranded conformation or under severe conformational stress. These differences in the alpha 2(1) chromatin structure were confirmed by the finding that the promoter was more accessible to restriction endonuclease digestion in the expressing tissues than in the nonexpressing tissues. Digestion of chromatin with Pst I and Sma I revealed that some of these sites in the promoter were differentially protected by DNA-binding proteins in the two tissue types. These protected sites were located as far upstream as -1,600 and downstream within the first intron at +800.

The abolition of collagen gene expression in SV40-transformed fibroblasts is associated with trans-acting factor switching.

The goal of this study was to determine whether alpha 2(1) procollagen gene expression is modulated by positive or negative trans-acting DNA-binding proteins. Previous studies have shown that a clone of SV40-transformed WI-38 fibroblasts (SVWI-38) does not produce any alpha 2(1) procollagen mRNA (Parker et al (1989), J. Biol Chem. 264, 7147-7152). In order to elucidate the mechanism(s) responsible for such inactivation, we have examined the activity of a transfected wild type COL1A2 promoter in SVWI-38 cells. A set of 5' promoter deletions was linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into SVWI-38 and other cell lines expression type I collagen. The resulting CAT assays confirmed the importance of several upstream regions for promoter activity and documented the decreased transcriptional activity from an exogenous COL1A2 promoter in the SVWI-38 cell line. Competition experiments with an excess of COL1A2 promoter DNA fragment and a constant amount of COL1A2/CAT construct displayed a linear relationship between excess COL1A2 fragment and CAT activity in SVWI-38 cells, suggesting the involvement of a titratable negative effector. Electrophoretic mobility shift assays revealed the presence of a specific DNA-protein complex which was present in SVWI-38 cells and almost absent in control fibroblasts. Methylation interference analysis mapped the region of binding of this factor between nucleotides -80 and -72, relative to the transcription start site. Thus the data presented provide strong evidence for the existence of a negative trans-acting factor that may play a role in the repression of COL1A2 expression in SVWI-38 fibroblasts.

Regulation of collagen I gene expression by ras.

Although transformation of rodent fibroblasts can lead to dramatic changes in expression of extracellular matrix genes, the molecular basis and physiological significance of these changes remain poorly understood. In this study, we have investigated the mechanism(s) by which ras affects expression of the genes encoding type I collagen. Levels of both alpha 1(I) and alpha 2(I) collagen mRNAs were markedly reduced in Rat 1 fibroblasts overexpressing either the N-rasLys-61 or the Ha-rasVal-12 oncogene. In fibroblasts conditionally transformed with N-rasLys-61, alpha 1(I) transcript levels began to decline within 8 h of ras induction and reached 1 to 5% of control levels after 96 h. In contrast, overexpression of normal ras p21 had no effect on alpha 1(I) or alpha 2(I) mRNA levels. Nuclear run-on experiments demonstrated that the transcription rates of both the alpha 1(I) and alpha 2(I) genes were significantly reduced in ras-transformed cells compared with those in parental cells. In addition, the alpha 1(I) transcript was less stable in transformed cells. Chimeric plasmids containing up to 3.6 kb of alpha 1(I) 5'-flanking DNA and up to 2.3 kb of the 3'-flanking region were expressed at equivalent levels in both normal and ras-transformed fibroblasts. However, a cosmid clone containing the entire mouse alpha 1(I) gene, including 3.7 kb of 5'- and 4 kb of 3'-flanking DNA, was expressed at reduced levels in fibroblasts overexpressing oncogenic ras. We conclude that oncogenic ras regulates the type I collagen genes at both transcriptional and posttranscriptional levels and that this effect, at least for the alpha 1(I) gene, may be mediated by sequences located either within the body of the gene itself or in the distal 3'-flanking region.

Elevation of large-T antigen production by sodium butyrate treatment of SV40-transformed WI-38 fibroblasts.

The effects of sodium butyrate on simian virus 40 early gene expression were determined in SV40-transformed human embryonic lung fibroblasts (SVWI-38). Northern blot analysis and nuclear run-off transcription studies revealed that treatment of cells with millimolar concentrations of sodium butyrate (2.5 to 10 mM) resulted in increased levels of SV40 early gene transcripts, with a concomitant increase in their corresponding proteins (large-T and small-t antigens). Although sodium butyrate treatment enhanced the expression of the early genes, it was associated with a reduction in cell growth and total protein synthesis, as measured by cell number and incorporation of 3H-leucine into macromolecules, respectively. Immunoprecipitation of 35S-labelled cellular proteins with anti-p53 and anti-T antibodies revealed that the level of the cellular protein, p53, declined markedly in the presence of sodium butyrate. Furthermore, in control cells only 30% of the p53 was complexed with large-T antigen, whereas in butyrate-treated cells all the p53 was complexed with large-T antigen. The increased early gene expression was not due to altered methylation patterns, gene amplification, or rearrangement of the integrated SV40 genome. Sodium butyrate treatment did, however, result in the appearance of a new nuclear protein which bound specifically to a SV40 promoter fragment containing large-T antigen binding sites I and II.

The genetic basis of cancer.

Cancer is essentially a genetic disease resulting from congenital or acquired alterations in some cells of the patient. Such changes may occur in particular oncogenes and are responsible for the tumour phenotype of the affected population of cells. Oncogenes function by continuous positive action in the mitogenic pathway, and may become activated by point mutations, chromosomal rearrangements, gene amplification or viral insertion events. In contrast, unaltered tumour-suppressor genes are responsible for suppressing the neoplastic phenotype, and their inactivation by deletion or mutation permits cancerous development in the affected cells. The genetic model of carcinogenesis is thus based on the idea that mutations at the DNA level create a functional imbalance between the oncogenes and the tumour-suppressor genes, resulting in uncontrolled clonal proliferation. It is likely that the clinical importance of these recent findings will soon be realised and utilised in the development of therapies and diagnostic procedures that will directly benefit the patient.

Is there a SV40 middle T antigen?

It is hypothesized that Simian Virus 40 (SV40) produces a middle T antigen with a mRNA almost the same size as the small t antigen mRNA and a protein similar in size to that of large T antigen. Both middle T mRNA and protein, therefore, would not be readily discernable. Disruption or removal of the small t antigen translation stop codon by post-transcriptional RNA processing could provide a mechanism to achieve the above. A key role for the small t intron in this process is suggested.