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Biomedical subjects

E K Parkinson

Publications and source records attributed to E K Parkinson.

At least 19 recordsLinked to original sources
PubMed2003

Human squamous cell carcinomas lose a mortality gene from chromosome 6q14.3 to q15.

Normal human keratinocytes possess a finite replicative lifespan. Most advanced squamous cell carcinomas (SCCs), however, are immortal, a phenotype that is associated with p53 and INK4A dysfunction, high levels of telomerase and loss of heterozygosity (LOH) at several genetic loci, suggestive of the dysfunction of other mortality genes. We show here that human chromosome 6 specifically reduces the proliferation or viability of a human SCC line, BICR31, possessing LOH across the chromosome. This was determined by an 88% reduction in colony yield (P<0.001), following the reintroduction of an intact normal chromosome 6 by monochromosome transfer. Deletion analysis of immortal segregants using polymorphic markers revealed the loss of a 2.9 Mbp interval, centred on marker D6S1045 at 6q14.3-q15, in 6/19 segregants. Crucially, allelic losses of this region were not identified in control hybrids constructed between chromosome 6 and the BICR6 SCC cell line that is heterozygous for chromosome 6 and which showed no reduction in colony formation relative to the control chromosome transfers. This indicates that the minimally deleted region at D6S1045 is not the result of fragile sites, a recombination hot spot, or a feature of the monochromosome transfer technique. LOH of D6S1045 was found in 2/9 immortal SCC lines and was part of a minimally deleted region of line BICR19. Furthermore, allelic imbalance, consistent with LOH, was detected in 3/17 advanced SCCs of the tongue. These results suggest the existence of a suppressor of SCC immortality and tumour development at chromosome 6q14.3-q15, which is important to a subset of human SCCs.

Carcinoma, Squamous Cell↗
PubMed2001

Detection of functional PTEN lipid phosphatase protein and enzyme activity in squamous cell carcinomas of the head and neck, despite loss of heterozygosity at this locus.

The human tumour suppressor gene PTEN located at 10q23 is mutated in a variety of tumour types particularly metastatic cases and in the germline of some individuals with Cowdens cancer predisposition syndrome. We have assessed the status of PTEN and associated pathways in cell lines derived from 19 squamous cell carcinomas of the head and neck. Loss of heterozygosity is evident at, or close to the PTEN gene in 5 cases, however there were no mutations in the remaining alleles. Furthermore by Western analysis PTEN protein levels are normal in all of these SCC-HN tumours and cell lines. To assess the possibility that PTEN may be inactivated by another mechanism, we characterized lipid phosphatase levels and from a specific PIP3 biochemical assay it is clear that PTEN is functionally active in all 19 human SCCs. Our data strongly suggest the possibility that a tumour suppressor gene associated with development of SCC-HN, other than PTEN, is located in this chromosomal region. This gene does not appear to be MXI-1, which has been implicated in some other human tumour types. PTEN is an important negative regulator of PI3Kinase, of which subunit alpha is frequently amplified in SCC-HN. To examine the possibility that PI3K is upregulated by amplification in this tumour set we assessed the phosphorylation status of Akt, a downstream target of PI3K. In all cases there is no detectable increase in Akt phosphorylation. Therefore there is no detectable defect in the PI3K pathway in SCC-HN suggesting that the reason for 3q26.3 over-representation may be due to genes other than PI3K110alpha.

Blotting, Western↗
PubMed2001

Human fibroblast replicative senescence can occur in the absence of extensive cell division and short telomeres.

Ectopic expression of telomerase blocks both telomeric attrition and senescence, suggesting that telomeric attrition is a mitotic counting mechanism that culminates in replicative senescence. By holding human fibroblast cultures confluent for up to 12 weeks at a time, we confirmed previous observations and showed that telomeric attrition requires cell division and also, that senescence occurs at a constant average telomere length, not at a constant time point. However, on resuming cell division, these long-term confluent (LTC) cultures completed 15-25 fewer mean population doublings (MPDs) than the controls prior to senescence. These lost divisions were mainly accounted for by slow cell turnover of the LTC cultures and by permanent cell cycle exit of 94% of the LTC cells, which resulted in many cell divisions being unmeasured by the MPD method. In the LTC cultures, p27(KIP1) accumulated and pRb became under-phosphorylated and under-expressed. Also, coincident with permanent cell cycle exit and before 1 MPD was completed, the LTC cultures upregulated the cell cycle inhibitors p21(WAF) and p16(INK4A) but not p14(ARF) and developed other markers of senescence. We then tested the relationship between cell cycle re-entry and the cell cycle-inhibitory proteins following subculture of the LTC cultures. In these cultures, the downregulation of p27(KIP1) and the phosphorylation of pRb preceded the complete resumption of normal proliferation rate, which was accompanied by the down-regulation of p16(INK4A). Our results show that most normal human fibroblasts can accumulate p16(INK4A), p21(WAF) and p27(KIP1) and senesce by cell division-independent mechanism(s). Furthermore, this form of senescence likely requires p16(INK4A) and perhaps p27(KIP1).

3T3 Cells↗
PubMed2001

Decreased expression of TGF-beta cell surface receptors during progression of human oral squamous cell carcinoma.

This study examined the immunocytochemical expression of the transforming growth factor-beta (TGF-beta) isoforms TGF-beta1, TGF-beta2, and TGF-beta3, together with the TGF-beta cell surface receptors TbetaR-I and TbetaR-II, in patient-matched tissue pairs of normal human oral epithelium, primary squamous cell carcinomas, and metastatic lymph node tumour deposits. There were no significant differences in the intensity of TGF-beta isoform specific staining between the normal oral epithelium, the primary tumours, and the lymph node metastases. By contrast, there was significantly less TbetaR-II in the metastases than in the primary tumour and between the primary tumour and the normal oral epithelium. Similar trends were evident with TbetaR-I, but not at a statistically significant level. This study also examined the structure of TbetaR-I and TbetaR-II in normal human oral keratinocytes in vitro and in 14 human oral carcinoma cell lines with known responses to TGF-beta1. No structural abnormalities of TbetaR-II were present in the normal keratinocytes or in 13 of 14 malignant cell lines; in one line, there were both normal and mutant forms of TbetaR-II, the latter being in the form of a frameshift mutation with the insertion of a single adenine base (bases 709-718, codons 125-128), predicting a truncated receptor having no kinase domain. No defects were present in TbetaR-I. The structures of TbetaR-I and TbetaR-II did not correlate with growth inhibition by TGF-beta1. The data suggest that decreased expression of TGF-beta receptors, rather than structural defects of these genes, may be important in oral epithelial tumour progression. In order to examine the functional significance of a specific decrease in TbetaR-II expression, a dominant-negative TbetaR-II construct (dnTbetaR-II) was transfected into a human oral carcinoma cell line with a normal TGF-beta receptor profile and known to be markedly inhibited by TGF-beta1. In those clones that overexpressed the dnTbetaR-II, growth inhibition and Smad binding activity were decreased, whilst the regulation of Fra-1 and collagenase-1 remained unchanged following treatment with TGF-beta1. The results demonstrate that a decrease in TbetaR-II relative to TbetaR-I leads to selective gene regulation with loss of growth inhibition but continued transcription of AP-1-dependent genes that are involved in the regulation of the extracellular matrix.

Adult↗
PubMed2000

E-cadherin at the cell periphery is a determinant of keratinocyte differentiation in vitro.

The origin of the signal for keratinocyte differentiation is still unknown. Here, we show that Ca(2+)- and density-induced translocation of E-cadherin, but not P-cadherin, is accompanied by induction of differentiation-specific proteins in cultured keratinocytes. Antibodies that artificially cluster cell-surface E-cadherin in low extracellular Ca(2+) also induce differentiation-specific proteins, implicating E-cadherin as a determinant of keratinocyte differentiation in vitro.

Cadherins↗
PubMed2000

Replicative senescence as a barrier to human cancer.

There is evidence that one critically short telomere may be recognized as DNA damage and, as a consequence, induce a p53/p21WAF- and p16INK4A-dependent G1 cell cycle checkpoint to cause senescence. Additionally, senescence via a p53- and p16(INK4A)-dependent mechanism can be induced by the over- or under-stimulation of certain signalling pathways that are involved in cancer. Central to this alternative senescence mechanism is the p14ARF protein, which connects oncogene activation, but not DNA damage, to p53 activation and senescence. We find that immortal keratinocytes almost invariably have dysfunctional p53 and p16 and have high levels of telomerase, but very often express a wild-type p14(ARF). Furthermore, when normal keratinocytes senesce they show a striking elevation of p16 protein, but not of p14(ARF) or its downstream targets p53 and p21(WAF). These results suggest that p16, rather than p14(ARF), is the more important gene in human keratinocyte senescence, but do not exclude a co-operative role for p14(ARF), perhaps in the induction of senescence by activated oncogenes in neoplasia. Regardless of mechanism, these results suggest that replicative senescence acts as a barrier to human cancer development.

Carrier Proteins↗
PubMed2000

The catalytic activity of the Src family kinases is required to disrupt cadherin-dependent cell-cell contacts.

Despite the importance of epithelial cell contacts in determining cell behavior, we still lack a detailed understanding of the assembly and disassembly of intercellular contacts. Here we examined the role of the catalytic activity of the Src family kinases at epithelial cell contacts in vitro. Like E- and P-cadherin, Ca(2+) treatment of normal and tumor-derived human keratinocytes resulted in c-Yes (and c-Src and Fyn), as well as their putative substrate p120(CTN), being recruited to cell-cell contacts. A tyrosine kinase inhibitor with selectivity against the Src family kinases, PD162531, and a dominant-inhibitory c-Src protein that interferes with the catalytic function of the endogenous Src kinases induced cell-cell contact and E-cadherin redistribution, even in low Ca(2+), which does not normally support stable cell-cell adhesion. Time-lapse microscopy demonstrated that Src kinase inhibition induced stabilization of transiently formed intercellular contacts in low Ca(2+). Furthermore, a combination of E- and P-cadherin-specific antibodies suppressed cell-cell contact, indicating cadherin involvement. As a consequence of contact stabilization, normal cells were unable to dissociate from an epithelial sheet formed at high density and repair a wound in vitro, although individual cells were still motile. Thus, cadherin-dependent contacts can be stabilized both by high Ca(2+) and by inhibiting Src activity in low (0.03 mM) Ca(2+) in vitro.

Actins↗
PubMed1999

Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells.

Focal adhesion kinase (pp125FAK) is present at sites of cell/extracellular matrix adhesion and has been implicated in the control of cell behaviour. In particular, as a key component of integrin-stimulated signal transduction pathways, pp125FAK is involved in cellular processes such as spreading, motility, growth and survival. In addition, a number of reports have indicated that pp125FAK may be up-regulated in human tumour cells of diverse origin, and consequently, a role has been proposed for pp125FAK in the development of invasive cancers. However, to date the mechanisms that lead to elevated pp125FAK expression in tumour cells have not been determined. Here we used in situ hybridization to confirm chromosome 8q as the genomic location of the human fak gene and report that elevation of pp125FAK protein in cell lines derived from invasive squamous cell carcinomas is accompanied by gains in copy number of the fak gene in all cases examined. In addition, we observed increased fak copy number in frozen sections of squamous cell carcinomas. Furthermore, increased dosage of the fak gene was also observed in many cell lines derived from human tumours of lung, breast and colon, including two cell lines Calu3 and HT29, in which fak was amplified. In addition, in an in vitro model for human colon cancer progression there was a copy number gain of the fak gene during conversion from adenoma to carcinoma, which was associated with increased pp125FAK protein expression. Thus, we show for the first time that many cell lines derived from invasive epithelial tumours have increased dosage of the fak gene, which may contribute to the elevated protein expression commonly observed. Although other genes near the fak locus are co-amplified or increased in copy number, including the proto-oncogene c-myc, the biological properties of pp125FAK in controlling the growth, survival and invasiveness of tumour cells, suggest that it may contribute to the selection pressure for maintaining increased dosage of the region of chromosome 8q that encodes these genes.

Adenoma↗
PubMed1999

Role of the alternative INK4A proteins in human keratinocyte senescence: evidence for the specific inactivation of p16INK4A upon immortalization.

The INK4A locus on human chromosome 9p21 encodes two genes that have been implicated in replicative senescence and tumor suppression, p16INK4A and p14ARF. In contrast to p16INK4A, which is up-regulated to high levels, we were unable to detect p14ARF protein in senescent human keratinocytes. Also, p53, an established target of p14ARF, did not increase, suggesting that p14ARF is not instrumental in human keratinocyte senescence. In neoplastic keratinocyte cultures, p16INK4A inactivation was invariably associated with the immortal phenotype, and there was evidence for the inactivation of p16INK4A, independent of p14ARF, in 6 of 10 lines that lacked large homozygous deletions. In contrast, we failed to detect exon 1beta mutations or p16INK4A-independent deletions. These results emphasize the previously proposed role for p16INK4A in human keratinocyte senescence but do not rule out a supporting role for p14ARF inactivation.

3T3 Cells↗
PubMed1999

Genetic and functional analyses exclude mortality factor 4 (MORF4) as a keratinocyte senescence gene.

Approximately 50% of immortal human keratinocyte lines show loss of heterozygosity of chromosome region 4q33-q34, and the reintroduction of chromosome 4 into one such line, BICR 6, causes proliferation arrest and features of replicative senescence. Recently, a candidate gene, mortality factor 4 (MORF4), was identified in this region and sequenced in 21 immortal keratinocyte lines. There were no mutations or deletions, and two of the seven lines that showed loss of heterozygosity at 4q33-q34 were heterozygous for MORF4 itself. Furthermore, the transfer of a chromosomal segment containing the entire MORF4 gene did not mimic the senescence effect of chromosome 4 in BICR 6. These results suggest that the inactivation of MORF4 is not required for human keratinocyte immortality.

Cell Line, Transformed↗
PubMed1999

Analysis of the CAVEOLIN-1 gene at human chromosome 7q31.1 in primary tumours and tumour-derived cell lines.

We identified CAVEOLIN-1 as a candidate for a tumour suppressor gene mapping to human chromosome 7q31.1. A number of studies suggest that caveolin could function as a tumour suppressor. Expression of caveolin, and in turn the number of caveolae within a cell, are inversely correlated with the transforming ability of numerous oncoproteins, including H-ras, v-abl, and bcr-abl, and caveolin is a major transformation-dependent substrate of v-src. Heterologous expression of caveolin has been shown to abrogate anchorage-independent growth and induce apoptosis in transformed fibroblasts and also to suppress anchorage-independent growth in human mammary carcinoma cells. We have analysed the status and expression of the human CAVEOLIN-1 gene in primary tumours and tumour-derived cell lines. We found no evidence for mutation of CAVEOLIN-1 in human cancers. Additionally, we found that while the first two exons of CAVEOLIN-1 are associated with a CpG island, this is not methylated in either primary tumours or in tumour-derived cell lines in which Caveolin-1 expression is low or undetectable. The level of expression of Caveolin-1 does not correlate with loss of heterozygosity at the CAVEOLIN-1 locus in these same cell lines. Contrary to other published studies, we have shown that CAVEOLIN-1 is not expressed in normal breast ductal epithelial cells in vivo. CAVEOLIN-1 is however highly expressed in breast myoepithelial cells and its expression is retained in tumours derived from breast myoepithelium. Together our data refute a role for CAVEOLIN-1 as a breast tumour suppressor gene in vivo.

Breast Neoplasms↗
PubMed1999

Telomerase repressor sequences on chromosome 3 and induction of permanent growth arrest in human breast cancer cells.

BACKGROUND: Activation of the enzyme telomerase, which has been associated with cellular immortality, may constitute a key step in the development of human cancer. Telomerase is repressed in most normal human somatic cells. This study was conducted, using a genetic complementation approach, with the aim of identifying and mapping the genes responsible for repressing telomerase and, simultaneously, to establish the effect of experimentally induced telomerase repression on human tumor cell growth. METHODS: Individual human chromosomes isolated from normal diploid cells and tagged with bacterial antibiotic resistance genes (for later selection) were introduced into cells of the human breast carcinoma cell line 21NT by means of microcell transfer. Selected hybrid clones were screened for telomerase activity by use of the polymerase chain reaction-based telomere repeat amplification protocol (TRAP) assay, and the proliferative fate of the hybrid clones was determined. Regions of the introduced chromosomes associated with telomerase repression were mapped using segregant hybrids and a deletion analysis that employed microsatellite DNA markers. RESULTS: Strong repression of telomerase was observed following transfer of human chromosome 3 into 21NT cells but not after transfer of chromosomes 8, 12, or 20. The vast majority of hybrid clones with repressed telomerase entered permanent growth arrest after 10-18 population doublings. Deletion analysis of nonrepressed segregant monochromosome 3 hybrids indicated two regions on the short arm of chromosome 3 (3p21.3-p22 and 3p12-21.1) where telomerase regulator genes may be located. CONCLUSIONS: Telomerase in human breast cancer cells is efficiently repressed by a gene or genes on normal human chromosome 3p, and this repression is associated with permanent growth arrest of the tumor cells.

Adult↗
PubMed1999

Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes.

Based on the dominance of cellular senescence over immortality, immortal human cell lines have been assigned to four complementation groups for indefinite division. Human chromosomes carrying senescence genes have been identified, including chromosome 4. We report the cloning and identification of a gene, mortality factor 4 (MORF 4), which induces a senescent-like phenotype in immortal cell lines assigned to complementation group B with concomitant changes in two markers for senescence. MORF 4 is a member of a novel family of genes with transcription factor-like motifs. We present here the sequences of the seven family members, their chromosomal locations, and a partial characterization of the three members that are expressed. Elucidation of the mechanism of action of these genes should enhance our understanding of growth regulation and cellular aging.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of human and mouse telomerase RNA gene promoter sequences.

Variation in telomerase activity is correlated with cellular senescence and tumour progression. However, although the enzymatic activity of telomerase has been well studied, very little is known about how expression of telomerase genes is regulated in mammalian cells. We have therefore cloned the promoter regions of the human (hTR), and mouse, (terc), telomerase RNA genes in order to identify the regulatory elements controlling telomerase RNA gene transcription. 1.76 kb encompassing the hTR gene promoter region was sequenced, as was 4 kb encompassing the terc promoter. No significant sequence similarity could be detected in comparisons between human and mouse 5'-regions, flanking the transcribed sequences. However, both the human and mouse telomerase RNA genes are within CpG islands and may therefore be under the regulation of DNA methylation. Transient expression of hTR-reporter gene constructs in HeLa and GM847 cells identified the elements responsible for promoter activity are contained in a 231 bp region upstream of the transcriptional start site. Transient expression of terc-reporter gene constructs in Swiss3T3 and A9 cells identified the elements responsible for promoter activity are contained in a 73 bp region upstream of the transcriptional start site. These studies have implications for novel transcription targeted cancer therapies.

Animals↗
PubMed1997

Evidence for the inactivation of multiple replicative lifespan genes in immortal human squamous cell carcinoma keratinocytes.

Human keratinocyte immortality is genetically recessive to the normal phenotype of limited replicative lifespan and appears to require the dysfunction of p53 and the cyclin D-Cdk inhibitor p16. In order to test for the inactivation of other candidate replicative lifespan genes in the immortal cells of human tumors, we developed a series of mortal and immortal keratinocyte cultures derived from neoplastic lesions of the head and neck which were amenable to molecular genetic analysis by the loss of heterozygosity (LOH) technique. The results indicate that keratinocyte immortalization in head and neck squamous cell carcinoma (SCC-HN) development involves the inactivation of at least two further pathways to senescence and four in all. Chromosomes 1, 4 and 7 carry genes representing immortality complementation groups C, B and D respectively and immortal keratinocytes showed LOH at either 4q32-q34 between D4S1554 and D4S171 (group B) or 7q31 (group D) but never 1q25 (group C). These results tentatively suggest that the genes responsible for the immortality complementation groups encode proteins on the same pathway to senescence. In addition, all of the immortal keratinocyte lines possessed high levels of telomerase activity and a suppressor of telomerase activity has been mapped to the short arm of chromosome 3p. Five out of eight lines showed LOH at 3p21.2-p21.3, a region which may carry a gene capable of suppressing SCC-HN telomerase. However, alternative mechanisms of telomerase reactivation were also suggested by our results. None of the above genetic alterations were seen in seven senescent neoplastic keratinocyte cultures. Other loci harbouring antiproliferative genes implicated in replicative lifespan showed few or no alterations and any alterations seen were additional to those described above.

Carcinoma, Squamous Cell↗
PubMed1997

Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer.

Telomere length is maintained by the enzyme, telomerase, which has been linked to cellular immortality and tumour progression. However, the reasons for the high levels of telomerase found in human tumours are unknown. We have mapped the human telomerase RNA gene, (hTR), to chromosome 3q26.3 and show the hTR gene to be amplified in four carcinomas, (2/33 cervix, 1/31 head and neck, 1/9 lung). In addition, increased copy numbers of the hTR locus was also observed in 97% of tumours. By in situ hybridisation, the histological distribution of high levels of hTR expression could be demonstrated in a lung tumour and its metastasis with hTR amplification. These results are the first report of genetic alterations involving a known component of telomerase in human cancer. Indeed, it is also the first report of the amplification of a specific locus within the chromosome 3q region frequently subject to copy number gains in human tumours. In addition, we also show for the first time the histological distribution of the RNA component of telomerase in human tumours.

Carcinoma↗