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S Goodison

Publications and source records attributed to S Goodison.

32 records · Page 2Linked to original sources
PubMed1997

Detection of telomerase activity in exfoliated cancer cells in colonic luminal washings and its related clinical implications.

Telomerase is a ribonucleoprotein capable of replacing telomeric DNA sequences that are lost at each cell division. Under normal circumstances, it is active in rapidly dividing embryonic cells and in stem cell populations but not in terminally differentiated somatic cells. Much attention has recently focused on the hypothesis that activity of this enzyme is necessary for cells to become immortal. This predicts that telomerase activity should be detectable in malignant cells and tissues but not in their normal counterparts, which slowly senesce and die. In accordance with this notion, telomerase activity has been reported in a wide range of malignancies, including those of the gastrointestinal tract, breast and lung. In the present study, we used a polymerase chain reaction (PCR)-based assay for telomerase activity, designated the "telomeric repeat amplification protocol (TRAP)', to examine initially 35 colonic carcinomas, their corresponding normal tissues and 12 inflammatory bowel disease (IBD) lesions. We detected strong enzyme activity in 32 (92%) of the 35 colon carcinomas while there was no activity in 30 (86%) of 35 matched normal colonic tissue specimens and only very weak activity in the remainder. Four of seven specimens of ulcerative colitis and two of five Crohn's disease lesions were negative, and the rest were only weakly positive. These results led us to examine whether telomerase could be detected in carcinoma cells exfoliated into the colonic lumen. We assayed lysates of exfoliated cells in luminal washings from colectomy specimens of 15 patients with colon carcinoma and nine with IBD. Telomerase activity was detected in washings from 9 (60%) of the 15 colon carcinoma cases but not in any from cases with IBD, suggesting that it can be a good marker for the detection of colon carcinoma, possibly even in non-invasively obtained samples.

Aged↗
PubMed1997

Telomerase activity in human gynaecological malignancies.

AIM: To evaluate whether increased telomerase activity can be clinically useful for detecting malignant cells in a variety of gynaecological specimens. METHODS: Telomerase activity was examined in frozen tissue samples of histologically confirmed lesions of the endometrium, ovary, and cervix. It was also assessed in exfoliated cells in cervical smears from patients with premalignant and malignant lesions and in ascitic fluid obtained from cases with malignant or non-malignant ovarian tumours. RESULTS: Solid tissues from carcinomas were telomerase positive in all specimens of endometrial (6/6) and cervical (6/6) origin, and in almost all from the ovary (12/13). Normal tissues from the cervix (0/5) and the ovary (0/5) were telomerase negative, but samples from normal endometrium were found to show telomerase activity, possibly due to the cyclical regenerative nature of this tissue. Conversely, dissociated cells in cervical smears from preneoplastic and frankly neoplastic lesions rarely showed detectable telomerase activity. Thus smears from patients with malignant tumours were only positive in one of two patients, whereas those from CIN-2 (0/5) and CIN-3 (1/17) lesions and from normal (0/10) samples were almost all negative. Telomerase activity was also scarcely detectable in cells obtained from ascitic fluid from patients with ovarian tumours. CONCLUSIONS: As in many other organs, telomerase activity is increased in solid tissue specimens from malignant tumours of the female reproductive tract, but it is not yet a reliable indicator of the presence of exfoliated cancerous or precancerous cells in clinical specimens from such lesions. Interpretation should be guarded until more extensive studies have been conducted. The data on solid tissues presented here confirm that activation of this enzyme is a major hallmark of the neoplastic process.

Ascites↗
PubMed1996

Telomerase activity in human breast cancer and benign breast lesions: diagnostic applications in clinical specimens, including fine needle aspirates.

We analysed telomerase activity in normal, benign and malignant breast tissues and in fine needle aspirates by a PCR-based assay. The tissue samples we used in this assay consisted of 20 cryostat sections, 10 microns thick, from each breast biopsy. This method was used to obtain effective extraction from small samples and to confirm the histological identity of the specimen by microscopical examination of serial sections. Fifty-two of 71 breast carcinomas were positive for telomerase activity, and the intensity of this was strong in most cases, whereas all 6 samples of normal breast tissue and 17 of fibrocystic disease were negative and only 1 of 15 fibroadenomas was positive. Invasive ductal carcinomas were more frequently positive than invasive lobular carcinomas. There was no correlation of telomerase activity with tumour size or the occurrence of lymph node metastasis. Evaluation of our assay system showed that a signal of telomerase activity was detectable in extracts from single cryostat sections (< 1 mm2) of a cancer specimen and from as few as 4 cells of a human breast cancer cell line. On the basis of the above data, we applied this assay to fine needle aspirates of breast lesions. Ten of 15 aspirates which had been cytopathologically diagnosed as cancer were strongly positive, while 26 of 29 benign aspirates were totally negative and the remaining 3 showed only borderline activity. In 3 cases, the telomerase result could have helped establish a diagnosis when the cytological observations were inconclusive. Our results indicate that this sensitive assay could become a useful new modality for supplementing microscopic cytopathology in the detection of cancer cells in small tissue biopsies and fine needle aspirates.

Autoradiography↗
PubMed1996

Semiquantitative Detection of Abnormal CD44 Transcripts in Colon Carcinomas by Reverse Transcription-Polymerase Chain Reaction Enzyme-linked Immunosorbant Assay (RT-PCR ELISA).

Background: Abnormal expression of CD44 variant RNA has been detected in a variety of human tumors and has been shown to be a potential diagnostic marker. To date, such analysis requires time-consuming gel electrophoresis, blotting, and autoradiographic procedures, and this approach may not be suitable for routine laboratory examinations. We have developed a rapid and semiquantitative reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR ELISA) method and used it to analyze CD44 expression in colon carcinoma tissues and exfoliated cancer cells in colon luminal washings. Methods and Results: RNA was extracted from sample cells and tissues and converted to cDNA. PCR amplification products, labeled by incorporation of digoxigenin-11-dUTP, were hybridized with biotinylated probes complementary to CD44 exon 12 or to exons in the standard portion (CD44s) of the gene. Hybridized DNA complexes were immobilized on streptavidin-coated microtiter plates, and the bound PCR products were detected with a peroxidase-conjugated antibody to digoxigenin. CD44-derived PCR products were quantified by absorbance of a chromogenic reaction. Elevated expression of CD44 variant exon 12 was detected initially by Southern blot analysis in all of the 9 colon carcinoma tissues, while weak expression was observed in only 3 of 9 normal mucosas. This tumor-related differential expression was confirmed by the newly developed PCR-ELISA method. Elevated expression of CD44 exon 12 was also detected in exfoliated colonic epithelial cells from 10 of 13 carcinoma cases but not in exfoliated cells from 4 patients with inflammatory bowel disease. Conclusions: Raised expression of CD44 variant exon transcripts can be detected reliably in colonic tumor tissue and in exfoliated colonic cancer cells by a semiquantitative RT-PCR ELISA method. This was shown to be as sensitive as conventional RT-PCR using chemiluminescent detection. Therefore, CD44-based RT-PCR ELISA could facilitate detection of neoplasia in clinical specimens including colon washings and naturally micturated urine.

Journal Article↗
PubMed1996

Accumulation of Immature Intron-containing CD44 Gene Transcripts in Breast Cancer Tissues.

Background: Disorderly expression of the CD44 gene is a characteristic feature of many common types of human malignancy. The explanation for the diversity, abundancy, and abnormally large size of the resulting transcripts is under investigation. Methods and Results: This study used reverse transcription-polymerase chain reaction (RT-PCR)/Southern blot hybridization to examine the expression of the CD44 gene in fresh tissue samples from 21 breast cancers and 11 matched non-neoplastic breast tissue specimens from the same patients. Using probes for several exons and for a noncoding region (intron 9) of the gene, it was found that the previously described elevated levels of CD44 transcripts in malignant tumors of this organ include many unusual, alternatively spliced isotypes as well as immature forms that retain this intron and probably several others. All of the exons that were tested were involved in the disorderly overexpression of this gene observed in all of the cancer tissues, and we were able to detect retention of the intron 9 sequence in 14 (67%) of the 21 tumor samples. In contrast, it was possible to detect signals in only 3 (27%) of the 11 samples from nonmalignant breast tissue with this probe, and these were extremely faint. Conclusions: These findings imply that there is a profound disorder in the regulation of production, splicing, and processing of CD44 pre-mRNA in breast cancer tissues comparable to that described in tumors of many other organs. The clinical implication of this information is that analysis of tissue samples containing borderline or suspected premalignant lesions for the presence of these molecular abnormalities, which appear to be characteristic of neoplasia, may in due course help assessment of individual patient prognosis and optimization of treatment.

Journal Article↗
PubMed1996

Detection of exfoliated carcinoma cells in colonic luminal washings by identification of deranged patterns of expression of the CD44 gene.

AIMS: To investigate whether colonic cancer cells exfoliated into the lumen of the organ can be detected by identification of their abnormal CD44 gene products. METHODS: Exfoliated cells were obtained by centrifugation of saline wash-outs of 27 surgically resected colon specimens obtained from 15 patients with carcinoma, seven with ulcerative colitis and five with Crohn's disease. After extracting cellular mRNA, amplification by the reverse transcription-polymerase chain reaction (RT-PCR) technique and analysis by Southern blot hybridisation was carried out to examine the levels and patterns of transcription of exons 11(v6), and 12(v7) and intron 9 of the CD44 gene. The transcription of these CD44 components was also examined by RT-PCR of snap-frozen solid tissue specimens from 11 of the above patients with colorectal carcinoma, seven with ulcerative colitis and five with Crohn's disease. RESULTS: Abnormal expression of exons 11(v6) and 12(v7) was detected in exfoliated cells from 11 (73%) of 15 patients with carcinoma, but not in any patients with inflammatory bowel disease (IBD). The retention of intron 9 in CD44 mRNA transcripts was detected in washings from four (27%) carcinoma specimens but not in washings from non-malignant specimens. It was confirmed that in solid tissue samples from the same carcinomas there was abnormal over-expression of numerous alternatively spliced CD44 species containing transcripts of exons 11 and 12 and retention of intron 9. Low level expression of these exons was detected in tissue from inflammatory lesions from five of seven patients with ulcerative colitis and four of five with Crohn's disease. The retention of intron 9 was not seen in normal mucosa nor IBD. CONCLUSION: Abnormal expression of the variant exons and of intron 9 of the CD44 gene in tumour cells exfoliated into the colonic lumen may be helpful markers for the early, non-invasive, diagnosis of colorectal cancer.

Aged↗
PubMed1996

Progressive loss of CD44 gene expression in invasive bladder cancer.

This study investigated CD44 gene expression at both the RNA and protein level in well differentiated superficial and in deeply invasive bladder carcinomas. Proteins were studied by immunohistochemistry using antibodies against standard (CD44s) and variant (CD44v) isoforms. mRNA was analyzed by reverse transcriptase polymerase chain reaction/Southern blotting and in situ hybridization. Immunostaining with antibodies against CD44s and CD44v2, -5 and -6 (exons 7, 10, and 11, respectively) showed that carcinoma cells in all papillary tumors expressed strong signals throughout the epithelium but especially in the basal layer, which abuts on the stroma. However, invasive tumors, which are believed to originate mainly from flat urothelial tumors or, less frequently, from papillary carcinomas, progressively lost CD44 proteins as they penetrated deeper and became less differentiated. This change was paralleled at the mRNA level, by the gradual loss of expression of CD44s and CD44v transcripts in deeply invasive tumors until they were virtually undetectable. Conversely, papillary tumors contained multiple higher molecular weight transcripts, suggesting that the loss of CD44 proteins in the more aggressive tumors is due to a disturbance in transcription. This concept was confirmed by in situ hybridization studies with a probe showing that substantial variant CD44 mRNA is located in the urothelium in papillary carcinomas but is absent from deeply invasive carcinomas. These results indicate that initially there is a striking increase in CD44 gene expression in early bladder carcinomas, relative to normal urothelium, but that this diminishes as the tumors acquire a more aggressive phenotype.

Base Sequence↗
PubMed1996

Analysis of anomalous CD44 gene expression in human breast, bladder, and colon cancer and correlation of observed mRNA and protein isoforms.

Many studies have now demonstrated disorganized overexpression of the CD44 gene in various types of human malignant tumors, and this abnormality has emerged as an interesting candidate marker for early cancer diagnosis. The purpose of this work was to analyze and compare the patterns of transcription and translation of this gene in human breast (ZR75-1; MDAMB-435 clone 4A4) and colon (HT29) tumor cell lines and in tumors of the breast, bladder, and colon, with the aim of identifying the most suitable analyte for diagnostic purposes. Transcription was studied by reverse transcription-polymerase chain reaction using CD44-specific primers and probes complementary to exons in the standard (exons 3 to 5 and 16 to 18) and variably expressed regions of this gene (exons 7, 8, 10, 11, and 15). Translation was investigated by Western blot analysis and immunohistochemistry using monoclonal antibodies specific to the standard form of CD44 and to the products of the same variant exons. Southern blot hybridization analysis of the reverse transcription-polymerase chain reaction products showed a large number of CD44 transcripts in tumor cells. Direct comparison of these Southern blots with Western blots on matched tumor-cell-line extracts indicated that most of the diverse mRNA isoforms did not detectably translate into proteins. However, immunohistochemistry of normal and malignant breast (n = 17 and 23, respectively), bladder (n = 5 and 19), and colon (n = 19 and 19) tissue specimens showed increased staining of CD44 standard and CD44 variant proteins in the carcinoma cells. Combination of this information with the data from reverse transcription-polymerase chain reaction and Western blot analysis indicates that the overexpression at the protein level involves only a minority of the aberrant RNA transcripts. We conclude that the development of methods for the accurate quantitation of over-abundant CD44 RNA species in clinical samples offers the most promising approach to improved early diagnosis of malignancy using this new marker.

Antibodies, Monoclonal↗
PubMed1995

Abnormal retention of intron 9 in CD44 gene transcripts in human gastrointestinal tumors.

We have recently identified a new exon of the CD44 gene and demonstrated abnormal retention of a noncoding section, intron 9, in mRNA from bladder carcinomas. To analyze this further, the present study examined CD44 gene expression in cell lines from 14 esophageal, 3 colonic, and 4 breast carcinomas and in fresh samples from 20 colorectal carcinomas and corresponding normal colonic mucosa, using reverse transcriptase followed by the polymerase chain reaction (RT-PCR). This confirmed that there was abnormal assembly of several exons of the gene in cell lines and in tumor tissues from these organs. However, the most striking new finding was that intron 9 was present in RNA from 11 esophageal, 3 colon, and 1 breast carcinoma cell line, respectively. This was confirmed by RNase and DNase digestion analysis. Moreover, it was detected both in nuclear and cytoplasmic mRNA fractions, indicating that abnormal splicing of pre-mRNA occurs in cancer cells. The abnormal retention of intron 9 in CD44 gene transcripts was also demonstrated in tumor tissues from 16 (80%) of 20 patients with colon carcinoma, but there was no correlation with Dukes' stage. The biological significance of these observations is not yet understood. However, it is clear that, as with the abnormal expression pattern of CD44 variant exons, intron 9 retention is a good-candidate molecular diagnostic tool for colorectal carcinomas.

Base Sequence↗
PubMed1994

Response to glucose of transcription factor binding to the rat insulin-1 gene promoter.

The mechanism by which glucose stimulates insulin gene expression has been investigated by studying the binding of nuclear proteins to a putative glucose-sensitive element (GSE) in the rat insulin-I gene promoter. Gel retardation assays showed that a specific binding activity was present in four different beta-cell lines. The binding activity was increased by glucose only in those beta-cell lines which were shown to retain glucose-regulated insulin gene transcription. However, a similar binding activity was also shown to be present in an alpha-cell line. The protein factor binding to the GSE was estimated to have a molecular weight of 27kD. This protein may play a pivotal role in glucose-regulated transcription of the insulin gene.

Animals↗
PubMed1992

Control of insulin gene expression by glucose.

Northern-blot analysis was used to demonstrate that an increase in extracellular glucose concentration increased the content of preproinsulin mRNA 2.3-fold in the beta-cell line HIT T15. A probe for the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase was used as a control. Mannoheptulose blocked this effect of glucose. A stimulatory effect on preproinsulin mRNA levels was also observed in response to mannose and to 4-methyl-2-oxopentanoate. However, galactose and arginine were ineffective. Glucagon, forskolin and dibutyryl cyclic AMP also elicited an increase in HIT-cell preproinsulin mRNA. The ability of the 5' upstream region of the preproinsulin gene to mediate the effect of glucose and other metabolites on transcription was studied by using a bacterial reporter gene technique. HIT cells were transfected with a plasmid, pOK1, containing the upstream region of the rat insulin-1 gene (-345 to +1) linked to chloramphenicol acetyltransferase (CAT). Co-transfection with a plasmid pRSV beta-gal containing beta-galactosidase driven by the Rous sarcoma virus promoter was used as a control for the efficiency of transfection; expression of CAT activity in transfected HIT cells was normalized by reference to expression of beta-galactosidase. Glucose caused a dose-dependent increase in expression of CAT activity, with a half-maximal effect at 5.5 mM and a maximum response of 4-fold. Mannoheptulose blocked this effect of glucose. Other metabolites (mannose, 4-methyl-2-oxopentanoate and leucine plus glutamine) were also able to increase insulin promoter-driven CAT expression, but galactose and arginine were ineffective. The stimulatory effect of glucose on CAT expression was not blocked by verapamil and was inhibited by increasing extracellular Ca2+ from 0.4 to 5 mM. Both dibutyryl cyclic AMP and forskolin caused an increase in insulin promoter-driven gene expression in the presence of 1 mM-glucose, but neither agent further increased the level of expression occurring in the presence of a maximally stimulating glucose concentration. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also increased insulin promoter-driven CAT expression in the presence of 1 mM-, but not 11 mM-glucose. Staurosporine blocked the stimulatory effect not only of PMA but also of glucose and of dibutyryl cyclic AMP. We conclude that the 5' upstream region of the insulin gene contains sequences responsible for mediating the stimulatory effect of glucose on insulin-gene transcription.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaloids↗