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.

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.

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.

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.

Activation and demethylation of the intracisternal A particle genes by 5-azacytidine.

Treatment of C3H 10T1/2 mouse embryo fibroblasts with the cytidine analogues 5-azacytidine and 5-aza-2-deoxycytidine causes altered gene expression and results in the manifestation of phenotypic changes and altered cell morphology. This includes the conversion of these cells to adipocytes, chondrocytes and myotubes. The effects of these analogues on intracisternal A particle (IAP) gene expression in mouse C3H 10T1/2 cells have been examined. Treatment with either 3 microM 5-azacytidine or 0.3 microM 5-aza-2-deoxycytidine for 24 h was associated with an immediate increase in IAP gene transcription, and with the subsequent appearance of IAPs in the cisternae of the cells 24 h after removal of the drug. Control cells contained no, or very few, IAPs and IAP mRNA. Analysis of the methylation status of the IAP genes, using the restriction endonucleases HpaII, MspI and HhaI, showed that these genes were already demethylated at the end of the 24-h treatment period. IAP gene transcripts were detectable even after a 16-h drug treatment period, at which stage the genes were not yet fully demethylated. After further growth in fresh medium for 90 h, the levels of IAP RNA started to decline, but the demethylated CpG sites were not yet remethylated. These results suggest the involvement of other factors, in addition to methylation, in the regulation of IAP gene expression. These drugs were found to have no stimulatory effect on several oncogenes examined in this study.

Absence of alpha 2(1) procollagen synthesis in a clone of SV40-transformed WI-38 human fibroblasts.

Normal diploid human embryonic lung fibroblasts (WI-38) produce type I collagen of chain composition [alpha 1(1)]2.alpha 2(1) together with small amounts of type III collagen. We have examined the synthesis and secretion of type I collagen in a clone of SV40-transformed WI-38 fibroblasts (SVWI-38). These cells produced only 20-25% of the total collagen synthesized by their normal counterparts, with no detectable synthesis of alpha 2(1) chains and deposited a type I trimer consisting of overmodified alpha 1(1) chains. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cyanogen bromide peptides of collagens isolated from cells cultured either in the presence or absence of alpha,alpha-dipyridyl revealed that this overmodification occurred along the entire length of the alpha 1(1) chains. Analysis of poly(A+) RNA by Northern blot analysis and total RNA by slot blot analysis using cloned type I procollagen cDNA probes revealed that no alpha 2(1) mRNA was present in the SVWI-38 cells. Primer extension of the RNA confirmed this finding. The SVWI-38 cells had a normal chromosome number, but contained 28 normal and 18 abnormal and marker chromosomes. Restriction mapping of the entire alpha 2(1) procollagen gene did not reveal any gross deletions or insertions within this gene, nor was the gene hypermethylated in the transformed cells, when compared with their normal counterparts. One interesting feature, however, was the fact that certain CpG dinucleotides in the alpha 2(1) gene were methylated in the normal as well as the transformed cells. These SV40-transformed WI-38 fibroblasts therefore do not produce any alpha 2(1) collagen chains due to transcriptional inactivation of their genes.

Cyclophosphamide induced DNA strand breaks in mouse embryo cephalic tissue in vivo.

Pregnant C3H mice were exposed to teratogenic doses of cyclophosphamide (CPA), on the 11th day after copulation. The effects of this agent on embryonal cephalic DNA strand breaks were assessed between 3 and 40 h after drug administration. Administration of 15, 30 and 60 mg CPA/kg body weight resulted in conversion of 23, 30 and 44% of the DNA to the single-stranded form, respectively. No detectable DNA damage was evident 3 h after drug administration, but after 6 h significant DNA damage had occurred, reaching a maximum after 9 h. However, no evidence of DNA strand breaks was present at 22, 30 and 40 h after CPA treatment, suggesting that these lesions had been repaired. These findings demonstrate that cephalic DNA damage induced by CPA in the developing mouse embryo occurs in a time and concentration dependent manner, and provide some insight into the kinetics of formation and removal of DNA strand breaks caused by CPA in vivo.

Differential effects of DNA synthesis inhibitors on DNA methylation in normal and transformed cells.

1-beta-D-Arabinofuranosylcytosine (ara-C) and aphidicolin are well known inhibitors of DNA synthesis, each one acting through a different mechanism. We have examined the effects of these compounds on DNA methylation in normal human embryonic lung fibroblasts (WI-38) as well as in their Simian Virus 40 (SVWI-38) and gamma radiation (CT-1) transformed counterparts. Analysis of the methylation status of the total genomic DNA in WI-38 cells revealed that hydroxyurea was the only drug which resulted in a significant increase in the 5-methylcytosine content under conditions where greater than 98% inhibition of DNA synthesis was achieved. Under the same conditions, all three drugs were capable of inducing hypermethylation in the SVWI-38 cells, whereas none of them showed any effect on the methylation status of the DNA in the CT-1 cells. In cells where a limited degree of replication was allowed to occur at drug concentrations resulting in 50% inhibition of DNA synthesis, a different pattern emerged. Under these conditions, DNA which was synthesized in the presence of either ara-C or aphidicolin was significantly hypermethylated in both the transformed cell lines, whereas hydroxyurea had no effect. In the normal WI-38 cells however, hydroxyurea was still the only drug which caused any significant hypermethylation. Different cells thus responded differently to these three agents, and the mere slowing down of DNA synthesis did not ipso facto lead to increased DNA methylation.

Effects of sodium butyrate on the synthesis and methylation of DNA in normal cells and their transformed counterparts.

The effects of various concentrations of sodium butyrate were examined on a normal embryonic lung fibroblast cell line (WI-38) and its two transformed counterparts, a simian virus 40-transformed line (SVWI-38) and a cell line transformed by gamma-irradiation (CT-1). The rate of thymidine incorporation into DNA was inhibited by 60-80% in the WI-38 cells, even at butyrate concentrations as low as 5 mM. The two transformed cell lines showed no inhibition of DNA synthesis, even at concentrations of 75 mM butyrate. Analysis of RNA and protein synthesis revealed that the former was inhibited by +/- 20% at 5-10 mM butyrate in the normal WI-38 cell line, while protein synthesis was not inhibited at these concentrations. The inhibition of RNA synthesis was not dose dependent up to butyrate concentrations of 20 mM, and protein synthesis was inhibited less than 15% at this concentration. None of these inhibitory effects was observed in the case of the SVWI-38 or CT-1 cell lines. Analysis of the 5-methylcytosine content of DNA that was labeled either prior to or during treatment with butyrate revealed an increased content of methylcytosine when compared with control cells. Both preexisting and newly synthesized DNAs were thus subject to hypermethylation. Although all three cell lines showed a dose-dependent hypermethylation of DNA, the extent of this methylation differed in the normal and transformed lines, as preexisting DNA was more methylated in WI-38 cells compared with SVWI-38 and CT-1 cells, while methylation of newly synthesized DNA occurred to a greater extent in the SVWI-38 cells. These studies show that sodium butyrate affects major macromolecular synthetic processes as well as DNA methylation quite differently in normal and transformed cells.

Maternal administration of cyclophosphamide induces chromosomal aberrations and inhibits cell number, histone synthesis, and DNA synthesis in preimplantation mouse embryos.

The effects of cyclophosphamide (CPA), administered to pregnant inbred CBA/Ca mice 60 h after copulation, on cell number, mitotic index, chromosome structure, histone synthesis, and DNA synthesis of 84-h blastocysts, and the subsequent development of these blastocysts cultured for a further 120 h in vitro are described. Cyclophosphamide 4, 20, and 40 mg/kg significantly increased the number of chromosomally aberrant cells, chromosomal aberrations, and chromosome breaks in the blastocysts. Chromosomal rearrangements were significantly increased in the CPA 20 and 40-mg/kg treated groups, and in the 40-mg/kg group the number of cells with ring chromosomes was significantly increased. Histone synthesis and DNA synthesis were significantly inhibited in the CPA 20 and 40-mg/kg treated groups. Blastocyst cell number in each of the treated groups was less than the controls. On subsequent culture in vitro, significantly fewer embryos in the CPA 20 and 40-mg/kg groups hatched, attached, developed trophoblast outgrowths, and expanded their inner cell masses. However, the differentiation of inner cell mass into ectoderm and endoderm was impaired by all three doses of the drug. These results demonstrate that CPA administered to pregnant mice 60 h after copulation has a clastogenic effect and interferes with synthesis of DNA and histones in the preimplantation embryo, and that the drug inhibits the subsequent development and differentiation of these embryos. Cytogenetic analysis of preimplantation embryos might be a useful adjunct to the existing methods in the evaluation of the embryotoxicity of drugs and chemicals.