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S A Burchill

Publications and source records attributed to S A Burchill.

At least 37 records · Page 2Linked to original sources
PubMed1997

EWS-FLI1 fusion transcripts identified in patients with typical neuroblastoma.

The t(11.22)(q24.q12) results in expression of a chimeric RNA product, EWS-FLI1. This RNA product is expressed in over 85% of tumours belonging to the Ewing's family, and is increasingly used as a definitive characteristic of these tumours. In this study, we evaluated reverse transcriptase-polymerase chain (RT-PCR) for EWS-FLI1 fusion transcripts in 18 neurally derived small round cell tumours. These included six Ewing's family tumours and 12 neuroblastomas. EWS-FLI1 fusion transcripts were identified in all six Ewing's tumours, but also in two of the 12 neuroblastomas. One neuroblastoma contained the classic type 1 fusion transcript, and the second a type 1 transcript containing a 66 bp (base pair) insert that was not derived from the EWS or FLI1 gene. The presence of EWS-FLI1 fusion products in RNA extracted from primary neuroblastoma suggests the identification of EWS-FLI1 fusion transcripts is not pathognomonic for tumours of the Ewing's family. The clinical significance of these fusion transcripts in neuroblastoma is not known.

Adolescent↗
PubMed1995

Activation of p21ras by nerve growth factor in neuroblastoma cells.

Nerve growth factor (NGF) is essential for the differentiation and survival of sympathetic and sensory neurones and is thought to play a role in the differentiation of neuroblastoma. In this study we have shown NGF decreased the mRNA level of the two GTPase activating proteins neurofibromin (containing the NF1-GRD) and type 1 GAP120 in two neuroblastoma cell lines, IMR-32 and SK-N-SH. This effect was seen within 15 min exposure to NGF and was maintained up to 2 h after the addition of NGF. Treatment with NGF increased the amount of GTP bound p21ras 3-fold, within 20 min exposure. Western blot analysis showed SK-N-SH and IMR-32 cells to contain equal amounts of p21ras protein and these levels were unchanged by NGF treatment. However, NGF induced an increase in the level of neurofilament L protein, which was accompanied by an increase in neurite extension. These effects of NGF occurred in the absence of growth inhibition. In conclusion, our results demonstrate a decrease in GTPase activating proteins and activation of p21ras by NGF in IMR-32 and SK-N-SH cells, thus implicating p21ras in NGF signal transduction in neuroblastoma.

Base Sequence↗
PubMed1995

Early clinical evaluation of neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase mRNA.

Disseminating disease in neuroblastoma is of considerable clinical importance. Detection of circulating neuroblastoma cells using tyrosine hydroxylase (TH) as a tissue-specific target for reverse transcriptase-polymerase chain reaction has proved to be a sensitive and specific method for the detection of contaminating tumour cells in peripheral blood. The aim of this study was to report the early clinical observations made using this technology in neuroblastoma patient blood samples. A strong association was found between the detection of neuroblastoma cells in circulation with the detection of neuroblastoma in bone marrow. This method may be of use to monitor disease status and identify early signs of relapse in clinically disease-free patients. These results show that RT-PCR detection of TH mRNA is a relatively noninvasive, sensitive method for the detection of circulating tumour cells in neuroblastoma patients.

Child↗
PubMed1995

Effect of retinoic acid on p21ras and regulators of its activity in neuroblastoma.

p21ras is a membrane-associated guanine nucleotide-binding protein with intrinsic GTPase activity. This protein is important in the regulation of cell growth and differentiation in a number of different cell types. Therefore, the aim of the present study was to examine the role of p21ras and regulators of its activity in the differentiation of neuroblastoma cells induced by retinoic acid (RA). Phosphorylation of p21ras is regulated by the GTPase activity of type I GAP120 and neurofibromin. RA-induced differentiation of the two neuroblastoma cell lines SK-N-SH and IMR-32 was closely related to growth inhibition. Differentiation induced by RA resulted in an increase in both type I GAP120 and neurofibromin mRNAs. This increase was accompanied by a decrease in the activation of p21ras. These results suggest that, in neuroblastoma, activation of p21ras is not associated with RA-induced differentiation. However, the GTPase activating proteins type I GAP120 and neurofibromin may have effector functions in RA-induced differentiation of neuroblastoma.

Base Sequence↗
PubMed1995

Detection of epithelial cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction.

Circulating cancer cells in the blood play a central role in the metastatic process. Their number can be very small and techniques for their detection need to be both sensitive and specific. Polymerase chain reaction (PCR) has been successfully used to detect small numbers of tumour cells in haematological cancer in which abnormalities in DNA are sufficiently consistent to make this possible. For most solid tumours this not yet feasible. However, we have found that reverse transcriptase (RT)-PRC for tissue-specific gene expression is a useful technique for identifying small numbers of circulating cells in melanoma and neuroblastoma patients. In this report we describe detection of colon carcinoma cells by RT-PCR using CK 20 mRNA as a marker. Unlike other cytokeratin genes examined (CK 8 and CK 19), CK 20 was not transcribed in normal haematopoietic cells. This suggests a role for RT-PCR in the detection of colon carcinoma metastasis in blood and bone marrow, using CK 20 as the target gene. Future analysis of clinical material will determine the clinical significance of this technique.

Adenocarcinoma↗
PubMed1994

Neuroblastoma cell detection by reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosine hydroxylase mRNA.

The presence of tumour cells in peripheral blood of neuroblastoma patients is of considerable clinical importance. Nucleic acid amplification offers an opportunity to detect very small numbers of such cells, but in neuroblastoma a frequent specific abnormality in the tumour DNA suitable for this purpose has yet to be identified. To facilitate the detection of such cells we have developed RT-PCR using tyrosine hydroxylase (TH) as a tissue-specific target gene. TH mRNA was detected in 3 neuroblastoma cell lines and in all neuroblastoma tumours examined, but was undetectable in peripheral blood from children without neuroblastoma. The method was highly sensitive, detecting 1-10 neuroblastoma cells per 10(7) blood cells. Thirty blood samples from 24 patients were analysed and results were compared with known disease status. At diagnosis 4/7 patient blood specimens were positive; the four positive samples were from stage-4 patients. In blood samples from these patients 6-8 weeks after the initiation of treatment, TH mRNA was undetectable. Of 7 samples taken at the time of clinical relapse, 5 were positive; 4 of these were from patients with evidence of disseminating disease. Of 16 blood samples from disease-free patients, 14 were negative and 2 were positive. One positive patient in this group subsequently had a clinical relapse. These results show that this technique is of value for detecting neuroblastoma cells in peripheral blood. The significance of these cells at diagnosis, during treatment or on follow-up requires further evaluation.

Base Sequence↗
PubMed1994

Changing expression of GTPase activating proteins with differentiation in neuroblastoma.

p21ras is a membrane-associated guanine nucleotide-binding protein with intrinsic GTPase activity. Like other guanine nucleotide-binding proteins p21ras is active when GTP bound and inactive when GDP bound. Phosphorylation of p21ras is regulated by the GTPase activity of type I GAP120 and NF1-GRD. In this study we have identified type I GAP120 and two NF1-GRD mRNAs in three neuroblastoma cell lines, IMR-32, SK-N-SH and SK-N-MC. NF1-GRD mRNA was expressed in all cell lines at a similar level but type I GAP120 mRNA was more abundant in the IMR-32 cell line. Retinoic acid induced differentiation of all three cell lines, this effect was most marked in the SK-N-SH line. This differentiation was accompanied by an increase in both type I GAP120 and NF1-GRD mRNAs. Retinoic acid induced differentiation had no effect on the ratio of type I to type II NF1-GRD mRNA. In seven patient tumour samples examined type I GAP120 and NF1-GRD were coexpressed, type I GAP120 at a higher level than NF1-GRD in all tumour stages. Type I was the predominant NF1-GRD mRNA. The expression of type I GAP120 was similar in all tumour stages but the total level of NF1-GRD was higher in stage 2 and 3 tumours than in stage 4 tumours. In summary, these results suggest increased type I GAP120 and NF1-GRD mRNA are associated with differentiation in neuroblastoma cells.

Base Sequence↗
PubMed1994

Frequency of H-ras mutations in human bladder cancer detected by direct sequencing.

OBJECTIVE: To determine the frequency of mutations of the H-ras gene in transitional cell carcinomas of the human urinary bladder using direct DNA sequencing based on the polymerase chain reaction (PCR) method and to compare the results with those of other methods. In addition, the relationship of the mutation frequency to tumour stage and grade was examined. PATIENTS AND METHODS: Bladder tumour samples, taken by cystoscopic resection from 50 patients with newly diagnosed transitional cell carcinoma of the urinary bladder, were analysed by PCR-based direct DNA sequencing for point mutations in the H-ras gene at codon 12. RESULTS: Point mutations were found in 9 of 50 tumours examined (18%). The most frequent mutation (8/9) was a G to T transversion converting GGC to GTC, which would result in a glycine to valine substitution. The remaining mutations was a G to A transition altering GGC to GAC, producing a glycine to aspartic acid substitution, which has not previously been reported in bladder cancer. In all tumour samples examined the wild-type allele (GGC) was also evident. Variation in the proportion of wild-type to mutated sequence was found within tumour samples. No relationship between mutations and tumor grade and stage was apparent. CONCLUSION: The frequency of H-ras mutations detected in this first large scale study using the highly sensitive and rapid PCR-based sequencing method was comparable to that reported by earlier studies with the nude mouse tumorigenesis variation of the 3T3 transfection assay. H-ras mutations can be early events in the development and progression of a significant proportion of human bladder cancer cases.

Base Sequence↗
PubMed1993

Effects of melanocyte-stimulating hormone on tyrosinase expression and melanin synthesis in hair follicular melanocytes of the mouse.

Tyrosinase mRNA, synthesis and activity were measured in the skin during the first 2 weeks of life of C3H-HeAvy mice. Tyrosinase mRNA levels were found to peak on days 3-4 and were followed by increases in tyrosinase synthesis and activity which peaked on days 6-7 and 7-8 respectively. These changes in tyrosinase expression were presumably associated with the growth of the first coat of hair that in neonatal C3H-HeAvy mice is yellow in colour as a result of the increased proportion of phaeomelanin. By the time hair growth had ceased there was no expression of tyrosinase at both mRNA and protein levels. Daily administration of alpha-melanocyte stimulating hormone (alpha-MSH) enhanced the expression of tyrosinase mRNA transcripts, tyrosinase synthesis and activity. The increase in tyrosinase activity paralleled the change in the amount of tyrosinase, suggesting that the primary action of alpha-MSH is to stimulate new synthesis of the enzyme. This induction of tyrosinase was associated with the growth of hair that was darker in colour than that of the controls and contained an increased proportion of eumelanin. This increase in eumelanin reflected a decrease in phaeomelanin content. It was concluded that, through its actions on the enzyme tyrosinase, alpha-MSH is able to switch the synthesis of phaeomelanin to that of eumelanin in hair follicular melanocytes.

Animals↗
PubMed1992

Tyrosinase and the regulation of coat color changes in C3H-HeAvy mice.

Hair follicular tyrosinase activity was measured during hair growth in neonatal, pubertal, and adult C3H-HeAvy mice that show differences in coat color as a result of changes in the synthesis of eumelanin and pheomelanin. Tyrosinase activity increased during hair growth in all mice but higher levels were found at puberty, when the mice grow a dark, eumelanin coat of hair, than during early and adult life, when the hair follicular melanocytes produce mainly pheomelanin. This suggests that tyrosinase is more important for the synthesis of eumelanin than that of pheomelanin. The increased tyrosinase activity associated with eumelanogenesis in the pubertal mice could not be explained by enhanced transcription of the tyrosinase gene or enzyme synthesis and appeared to be the result of a post-translational activation. Such an activation of tyrosinase was lacking in the neonatal and adult mice; in the latter this was accompanied by a reduction in the glycosylation of tyrosinase and the proportion of enzyme associated with the melanosomal fraction. Our findings suggest that post-translational mechanisms are important control points in the regulation of tyrosinase and that differences in their level of activation are responsible for determining the patterns of melanogenesis in the C3H-HeAvy mice, but it is still not clear how these mechanisms are regulated. Although cyclic AMP increased tyrosinase synthesis it had no post-translation activating effect. The neonatal mice, unlike their pubertal and adult counterparts, also lacked dopachrome converting activity and TRP tyrosinase-related protein-1 expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Regulation of tyrosinase in hair follicular melanocytes of the mouse during the synthesis of eumelanin and phaeomelanin.

Tyrosinase activity, synthesis, mRNA, and its posttranslational processing were compared in hair follicular melanocytes of the C3HHeAvy mouse during eumelanogenesis and phaeomelanogenesis. Tyrosinase activity was increased during eumelanogenesis; this increase was accompanied by an increase in tyrosinase synthesis. Tyrosinase activity was also increased during phaeomelanogenesis, but only to a peak level that was 50% of that during eumelanogenesis. However, tyrosinase synthesis and mRNA levels were the same in follicles during eumelanin and phaeomelanin synthesis. The lower level of tyrosinase activity is, therefore, presumably due to posttranslational regulation. Less tyrosinase was associated with the particulate fraction during phaeomelanogenesis than during eumelanogenesis. Glycosylation of tyrosinase during phaeomelanogenesis was also reduced and may be the mechanism of control. Bromo-adenosine 3,5-cyclic monophosphate sodium salt increased glycosylation in both eumelanin and phaeomelanin, producing follicles; but this did not result in an increased uptake of tyrosinase onto the melanosome membrane. Therefore, although cAMP increased glycosylation of tyrosinase, the uptake of tyrosinase by the melanosome membrane appeared to be regulated by other systems that are limiting during phaeomelanogenesis, resulting in a lower level of tyrosinase activity.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Tyrosinase expression and its relationship to eumelanin and phaeomelanin synthesis in human hair follicles.

Tyrosinase expression was examined in hair follicles from twenty three red- and dark-haired individuals. Tyrosinase activity was greater in the hair follicles of red-haired subjects than in those from dark-haired subjects. Tyrosinase synthesis was also greater in the red-haired subjects and this presumably accounted for their higher levels of tyrosinase activity. The levels of tyrosinase synthesis in the red-haired subjects correlated well with the phaeomelanin content in the hair but in the dark-haired individuals a better correlation was seen with eumelanin. alpha-Melanocyte stimulating hormone failed to increase tyrosinase synthesis in the hair follicles of either group of subjects and in the follicles from the redheads actually produced a decrease. 8-Bromo-cyclic AMP, on the other hand, increased tyrosinase synthesis but only in the hair follicles from dark-haired subjects. These findings contrast with those previously reported in mice and it would appear that the control mechanisms that regulate tyrosinase in human melanocytes are different in many respects from those in mice.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Pheomelanin as well as eumelanin is present in human epidermis.

There are two types of melanin in mammals, the brownish black eumelanin and the reddish yellow pheomelanin. Eumelanin and pheomelanin are present in human hair and this study was carried out to see whether both pigments are also present in human epidermis. Samples of epidermis were obtained from suction blisters raised in the upper arm of 13 Caucasian subjects of skin types I, II, and III and analyzed for both eumelanin and pheomelanin using a procedure involving high-performance liquid chromatography. Eumelanin and pheomelanin were found in all epidermal samples and their relative proportions correlated well with those found in samples of hair taken from the same subjects. The lowest concentrations of eumelanin were found in subjects of skin type I, with higher levels in skin types II and III. The concentrations of pheomelanin were more variable and showed no relationship to skin type. Increases in the concentrations of both pigments occurred following PUVA therapy, but whereas the largest increases in eumelanin were seen in skin types II and III, the increases in pheomelanin showed little relationship to skin type. Unlike eumelanin, epidermal pheomelanin also showed little relationship to PUVA-induced tanning. The present findings could be particularly significant in view of recent suggestions that pheomelanin, rather than protecting the skin against UV radiation, may actually contribute to UV-induced skin damage.

Epidermis↗
PubMed1991

Tyrosinase expression and melanogenesis in melanotic and amelanotic B16 mouse melanoma cells.

Tyrosinase activity, abundance, and mRNA transcription were examined in three sublines of the B16 mouse melanoma. Tyrosinase activity and melanin content were highest in the B16-F1 cells, slightly less in the B16-F10, and markedly lower in the B16-F10-DD cells. No differences in the level of tyrosinase mRNA or protein were found in the three different sublines. Thus, the differences in tyrosinase expression arise from the post-translational modification of the enzyme causing its activation or inhibition.

Animals↗
PubMed1990

Tyrosinase synthesis in different skin types and the effects of alpha-melanocyte-stimulating hormone and cyclic AMP.

Tyrosinase synthesis and its regulation in human melanocytes was studied by measuring the incorporation of [35S] methionine into incubated skin biopsies. Tyrosinase was detected in all skin samples with the highest levels in skin type IV and the lowest levels in skin type I. Following psoralen ultraviolet A (PUVA) therapy for several weeks, significant increases in the amounts of tyrosinase were found in skin types III and IV. The presence of alpha-melanocyte-stimulating hormone (alpha-MSH) (100 mumol/l) or the long-acting analogue [Nle4, DPhe7] alpha-MSH (1-10 mumol/l) in the incubation medium failed to alter tyrosinase levels in the skin biopsies taken from patients both before and after receiving PUVA therapy. Bromo-adenosine 3,5-cyclic monophosphate sodium salt (8-bromo-cAMP) (10 mmol/l), on the other hand, increased the amounts of tyrosinase both before and after PUVA, but these effects were only seen in biopsies of type III and IV skin. These results indicate that MSH fails to stimulate tyrosinase synthesis in human melanocytes. Nevertheless, tyrosinase synthesis and its regulation by cyclic AMP-dependent mechanisms could be important control points in the pigmentary response.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Regulation of tyrosinase synthesis and its processing in the hair follicular melanocytes of the mouse during eumelanogenesis and phaeomelanogenesis.

Tyrosinase synthesis and its posttranslational processing was compared in hair follicular melanocytes of C3H-HeAvy mice during eumelanogenesis and phaeomelanogenesis. Tyrosinase activity was increased during eumelanogenesis and this was paralleled by an increase in tyrosinase synthesis, as measured by the incorporation of [35S]methionine. Although tyrosinase activity was lower during phaeomelanogenesis there was no change in tyrosinase synthesis. alpha-Melanocyte stimulating hormone (alpha-MSH) increased tyrosinase activity and its synthesis during eumelanogenesis but not during phaeomelanogenesis. Bromo-adenosine 3,5-cyclic monophosphate sodium salt (8-bromo-cAMP) was similarly effective during eumelanogenesis, but unlike alpha-MSH stimulated tyrosinase synthesis during phaeomelanogenesis. This suggests that during phaeomelanogenesis the melanocytes may fail to express MSH receptors. This cannot account for the lower tyrosinase activity, however, for alpha-MSH acts predominantly at the level of tyrosinase synthesis and this was similar during eumelanin and phaeomelanin production. The reduced tyrosinase activity is, therefore, presumably due to some posttranslational change. Accordingly, less tyrosinase was associated with the melanosomal fraction during phaeomelanogenesis than during eumelanogenesis. Glycosylation of tyrosinase, as measured by the incorporation of [3H]glucosamine was also reduced during phaeomelanogenesis. Although 8-bromo-cAMP increased glycosylation of tyrosinase in both eumelanin and phaeomelanin producing melanocytes, it failed to alter the subcellular distribution of the enzyme. It would appear that, although glycosylation of tyrosinase is lower during phaeomelanogenesis, the reduced tyrosinase expression is the result of decreased uptake of tyrosinase by the melanosome.

8-Bromo Cyclic Adenosine Monophosphate↗