HPV-16 and cervical intraepithelial neoplasia.
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Female↗
Biomedical subjects
T Crook
Publications and source records attributed to T Crook.
Clonal p53 mutation in primary cervical cancer: association with human-papillomavirus-negative tumours.
Analyses of cancer cell lines and of anal cancers suggest an inverse correlation between infection with human papillomavirus (HPV) and somatic mutation of the p53 tumour-suppressor gene. We have investigated this association in primary cervical tumours. Tumour-tissue samples from 28 women with primary cancer of the cervix were analysed for presence of HPV sequences and for somatic mutations of the p53 gene. Southern blot analysis and the polymerase chain reaction (PCR) showed that 25 of the tumours contained HPV sequences; 20 were HPV16 positive and 5 HPV18 positive. 17 tumours subjected to restriction fragment length polymorphism analysis for the short arm of chromosome 17 showed no evidence of allelic deletion. Sequencing of the entire coding region of the p53 gene by asymmetric PCR detected heterozygous point mutations in only 3 HPV-negative tumours. By contrast, in 21 HPV-positive cancers the p53 sequence was wild-type throughout. Our data indicate that loss of wild-type p53 function is important in the pathology of cervical cancer and that in the absence of an HPV-encoded gene product that mediates loss of p53 function, somatic mutation of the gene is required. This pattern of p53 mutation may partly explain the apparently worse prognosis of HPV-negative cervical cancers.
Adenocarcinoma↗
Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription.
The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half-life of the p53 protein in non-transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half-lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half-life was found to be similar to that in non-transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2- and 4-fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co-transfection with plasmids expressing the wild-type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Properties of p53 mutations detected in primary and secondary cervical cancers suggest mechanisms of metastasis and involvement of environmental carcinogens.
Primary human papillomavirus (HPV) positive anogenital cancers normally develop without somatic mutation within the p53 gene. In this study, however, we have identified p53 point mutations in metastases arising from HPV positive cervical carcinomas, suggesting that acquisition of p53 mutation may play a role in the progression of some HPV associated primary cancers. p53 mutants identified in anogenital cancers exhibit a dominant transforming phenotype and increased resistance to HPV16 E6 directed degradation. The association of p53 mutation with metastases may explain the poor prognosis reported for HPV negative primary cancers, many of which already contain mutant p53. A high proportion of p53 mutations detected in both primary and metastatic cancers are GC-->TA transversions, strongly suggesting a role for external carcinogens in the development of these cancers.
Animals↗
Lack of immortalizing activity of a human papillomavirus type 16 variant DNA with a mutation in the E2 gene isolated from normal human cervical keratinocytes.
The oncogenic potential of a human papillomavirus type 16 (HPV16) variant cloned from normal human cervical keratinocytes has been tested in vitro using primary rodent epithelial cells and human cervical keratinocytes. The HPV16 variant was able to extend the lifespan of, but failed to immortalize, human keratinocytes. It could however cooperate with an activated ras oncogene to transform primary rodent cells. Radioimmunoprecipitation assays of the rodent cells showed that they expressed the E7 protein. DNA sequence analysis of the URR/E6/E7 and E5 regions of the HPV16 showed them to be fully functional, but a deletion in the viral E2 open reading frame was detected. This truncated E2 only weakly stimulated transcription of the viral regulatory region. Complementation assays using the HPV16 variant and a full-length E2 enabled the cloned variant to immortalize human cervical keratinocytes with wild-type efficiency. These results suggest that other viral gene products in addition to E6/E7 may play an important role in the in vitro immortalization of cervical keratinocytes in HPV16 and the development of cervical cancer.
Amino Acid Sequence↗
Effects of phosphatidylserine in Alzheimer's disease.
We studied 51 patients meeting clinical criteria for probable Alzheimer's disease (AD). Patients were treated for 12 weeks with a formulation of bovine cortex phosphatidylserine (BC-PS; 100 mg t.i.d.) or placebo, and those treated with the drug improved on several cognitive measures relative to those administered placebo. Differences between treatment groups were most apparent among patients with less severe cognitive impairment. Results suggest that phosphatidylserine may be a promising candidate for study in the early stages of AD.
Aged↗
Noradrenergic intervention in Alzheimer's disease.
We report results of a double-blind, 13-week trial of the alpha2 noradrenergic agonist guanfacine in 29 patients meeting clinical criteria for probable Alzheimer's disease (AD). Patients studied were relatively young (mean = 71 years), well educated (mean = 13.5 years), and in the early stages of cognitive deterioration (mean Mini-Mental State score = 22). We found no evidence of improvement with a single .5-mg daily dose of guanfacine and some evidence of a modest negative effect of the drug on neuropsychological tests. We concluded that noradrenergic intervention alone is unlikely to be effective in Alzheimer's disease.
Aged↗
Degradation of p53 can be targeted by HPV E6 sequences distinct from those required for p53 binding and trans-activation.
Human papillomavirus (HPV) types 16 and 18 appear to play a role in the development of ano-genital malignancies, whereas HPV 6 and 11 are usually associated with benign lesions. One HPV16 oncoprotein, E6, complexes with and promotes degradation of the cellular tumor suppressor p53. Here we show that E6 proteins of both oncogenic and benign HPV types associate in vitro with p53, but binding by E6 proteins of benign HPV types cannot target p53 for degradation. A C-terminal region of E6 conserved among all HPV types is important for p53 binding. However, N-terminal sequences of E6 conserved only between oncogenic HPV types are necessary to direct p53 degradation. p53 binding by E6 appears necessary but not sufficient for this activity. All E6 proteins tested showed comparable transcriptional trans-activating activity, a property that does not require the ability to bind or direct degradation of p53.
Amino Acid Sequence↗
Anti-sense phosphorothioate oligonucleotides have both specific and non-specific effects on cells containing human papillomavirus type 16.
A range of specific nuclease resistant phosphorothioate oligodeoxynucleotides (S-oligos) complementary to mRNA of human papillomavirus type 16 (HPV16), were tested for their ability to inhibit cell proliferation and to alter the level of HPV-specific mRNA and proteins in CaSki cells, a human cervical carcinoma cell line containing HPV16 DNA. Only certain of the S-oligos to the viral upstream regulatory region (URR) and the early viral open reading frames (ORF), E6 and E7, were found to display any activity on the cells. These S-oligos were found to exhibit potent anti-proliferative activity at concentrations between 0.25 microM and 20 microM, inhibiting the uptake of [3H]-thymidine into CaSki cells by up to 90% at higher concentrations. The rate of synthesis of E6 and E7 proteins and the steady state level of the E7 protein however remained largely unchanged. E7 protein exhibited a greater decrease in phosphorylation in the presence of only one of the antisense oligos. Other S-oligos including a random sequence, unmodified sequences or O-methylphosphonate modified oligos, had no specific effect on the cells. The results imply that the anti-sense S-oligonucleotides had both specific anti-HPV16 and other non-specific effects on cell proliferation and synthesis of virally encoded proteins.
Base Sequence↗
p53 is frequently mutated in Burkitt's lymphoma cell lines.
A panel of 12 Burkitt's lymphoma cell lines and four other B cell lines were tested for the presence of mutations in p53. Protein analysis using a mutant-specific antibody and sequencing of both cDNA and genomic DNA revealed changes relative to the standard p53 protein sequence in 12 of the 16 lines studied, including 10 of the BL lines. Mutation of p53 in the BL lines was usually accompanied by loss of the other allele of p53. Testing of the mutated p53 cDNAs for gain of transforming activity or loss of growth suppression activity showed that several of the BL mutants were functionally altered from wild-type p53.
Alleles↗
Expression of RB and p53 proteins in HPV-positive and HPV-negative cervical carcinoma cell lines.
Mutations within the tumor suppressor genes Rb-1 and p53 are commonly found in many human malignancies, and loss of wild-type function of both p53 and RB appear to be important events in the development of these malignancies. Interference with normal RB and p53 function in the cell has apparently also been exploited by the oncogenic genital human papillomaviruses (HPVs), which encode transforming proteins capable of binding cellular RB and p53 proteins. We have investigated the expression of RB and p53 in a series of eight cervical carcinoma cell lines, six of which contain HPV sequences and two of which have arisen apparently independently of HPV infection. In the six HPV-positive lines, no evidence of abnormal RB or p53 protein could be detected. However, there was evidence for abnormal RB and p53 in the two HPV-negative lines. These data are consistent with the hypothesis that loss of wild-type RB and p53 function is necessary for tumor development and that such loss can occur either by mutation within the cellular gene or by expression of viral proteins capable of complexing wild-type cellular proteins.
Carcinoma↗
Proposed factor structure of the Geriatric Depression Scale.
The Geriatric Depression Scale (GDS) is commonly used to measure depression in the elderly. However, there have been no reports of the underlying structure of the GDS. To this end, the GDS was administered to 326 community-dwelling elderly subjects, and the data were subjected to a factor analysis. A five-factor solution was selected and, after a varimax rotation, the factors that emerged could be described as: (1) sad mood, (2) lack of energy, (3) positive mood, (4) agitation, and (5) social withdrawal. This solution accounted for 42.9% of the variance. Knowledge of the factor structure should aid both clinicians and researchers in the interpretation of responses on the GDS.
Activities of Daily Living↗
Modulation of immortalizing properties of human papillomavirus type 16 E7 by p53 expression.
The E7 protein is one of the principle transforming proteins encoded by human papillomavirus type 16 (HPV16), a virus strongly associated with the development of cervical carcinoma. In the present study we show that cotransfection of wild-type human or murine p53 sequences with E7 and ras markedly reduces transformation in baby rat kidney cells, although no effect of p53 is seen on the ability of E7 to transform an established mouse line to anchorage independence. In contrast, expression of mutant p53 strongly potentiates the transforming function of E7 and confers marked growth factor independence to cells cotransformed by E7 and ras. These data suggest that E7 and p53 function in separate yet complementary biochemical pathways.
Animals↗
p53 point mutation in HPV negative human cervical carcinoma cell lines.
Clinical and experimental evidence is consistent with a key role for transforming human papilloma viruses (HPVs) in the aetiology of anogenital carcinoma. Cervical carcinoma does, however, occasionally occur in the absence of HPV sequences (Riou et al., 1990). We have used a direct cDNA/PCR sequencing protocol to analyse the sequence of p53 mRNA expressed by HPV positive and negative cervical carcinoma cell lines. Six cell lines which contain HPV sequences express p53 mRNA which has wild-type sequence throughout conserved boxes 2, 3, 4 and 5. The two HPV negative cell lines (C33a and HT3) express mutant p53 mRNA. In each case the mutation occurs in an evolutionarily conserved amino acid. Our data suggest that loss of wild-type p53 function is important in development of cervical carcinoma, and that this might be achieved either by mutation within the p53 gene or the presence of a virally encoded p53 binding protein.
Base Sequence↗
Status of c-myc, p53 and retinoblastoma genes in human papillomavirus positive and negative squamous cell carcinomas of the anus.
We have examined a series of squamous cell carcinomas (SCC) of the anus, anal intraepithelial neoplasia grade III (AINII) lesions and haemorrhoids for the presence of sequences from transforming human papillomavirus (HPV) types by polymerase chain reaction (PCR)/Southern blotting. In addition, the same DNAs have been analysed for abnormalities in the c-myc, p53 and retinoblastoma (Rb-1) gene loci by Southern blotting. HPV16 sequences were detected in a total of 38 of 50 (76%) and HPV18 sequences in 4 of the 50 cancers (8%). Of 12 haemorrhoids examined, none contained HPV16 or HPV18 sequences. Amplification of c-myc was demonstrated in 15 of the 50 cancers (30%), of which 13 were HPV16 positive, and one also positive for HPV18. Amplification of c-myc was not observed in the 5 AINIII or any of the 41 haemorrhoid DNAs analysed. Rearrangement of c-myc was not seen in any of the DNAs. Gross rearrangement, or loss of p53 or Rb-1 loci was not observed in any normal or tumor tissue. However, in preliminary analysis of p53 sequence, three tumours negative for HPV were heterozygous for p53 point mutation whereas six HPV positive tumours and two haemorrhoids were wild-type sequence throughout exons four to ten.
Anus Neoplasms↗
Detection of novel splicing patterns in a HPV16-containing keratinocyte cell line.
The W12 cell line was derived from a low grade cervical lesion, and is unique among HPV16-containing cell lines in carrying its HPV16 genome as a multicopy episome. As such it is thought to be more representative of a premalignant HPV16-induced tumor than the cervical cancers from which other cell lines have been derived. Using the polymerase chain reaction (PCR), we report here the identification and cloning of a number of novel cDNA species, which appear to be characteristic of the W12 cell line. Two species were identified with E6* coding capacity (E6*I and E6*III). The smaller of these (1009 bp) was predicted to encode a novel E6*III polypeptide containing C-terminal amino acids derived from an out of frame region of the E2/E4 ORFs. The larger species (1480 bp) contained, in addition to the E6*I ORF, an intact E7 ORF and probably represents the transcript for E7 expression, as the E7 protein was readily detectable in the W12 cell line. Both species appeared to be transcribed from the p97 promoter which has been shown to be active in other cell lines. A putative E2 repressor cDNA (891 bp), an E1/E4 message (883 bp), and two novel late cDNA species (1757 and 2031 bp) were also detected, allowing the identification of a splice acceptor immediately in front of the L1 open reading frame (nt 5637) and a splice donor at nt 3631. Although the 1757-base species has the capacity to encode a full-length L1 protein, both messages use a splice donor at nt 1301, and are thus not analogous to late species previously identified in HPV11. Of the six cDNAs cloned, only the 1480-bp E7 message has been observed in other HPV16-containing cell lines. The presence of L1 transcripts, and an E2 repressor mRNA, although unexpected, may reflect the different origins of the W12 cell line.
Amino Acid Sequence↗
Impaired facial recognition memory in aging and dementia.
Young normals, aged normals, and patients with early and advanced probable dementia of the Alzheimer type (DAT) were administered a facial recognition memory task. A continuous recognition paradigm was used, in which subjects were instructed to identify the repeated faces in an ongoing series of faces presented on a video monitor screen. A signal detection analysis of the data revealed that the DAT patients were markedly impaired in their ability to discriminate between new and repeated faces. Multiple presentations of faces improved the recognition accuracy of the early DAT patients only, but their rate of learning was slower than that of the normal subjects. In comparison to the young normals, elderly normals exhibited a mild deficit in recognition memory. All of the elderly subject groups exhibited a more liberal response bias than the young normals, which eliminates the possibility that the impaired memory task performance of the aged subjects could be attributed to a more conservative test-taking strategy. The DAT patients exhibited impaired recognition even when the second presentation of a face immediately followed the first, which perhaps implies that task performance was also sensitive to the effect of DAT on visuoperceptual abilities or psychomotor speed.
Adolescent↗
Alterations in growth properties of human papilloma virus type 16 immortalised human cervical keratinocyte cell line correlate with amplification and overexpression of c-myc oncogene.
Clinical and epidemiological data support a role for human papilloma virus (HPV) type 16 in the pathogenesis of cervical carcinoma. The W12 cell line contains HPV16 sequences, predominantly as a high copy number episome, and is immortalised in vitro, but non-tumourigenic. A morphologically distinct sub-line of the parental W12 cell line was isolated which displayed increased growth rate, increased capacity for self-renewal and increased resistance to differentiation in comparison with the parental W12 cell line. The structure and expression of the HPV16 sequences was virtually identical in the two cell lines. However, expression of the c-myc proto-oncogene was elevated in the morphologically distinct sub-line, and this was associated with amplification, but not rearrangement, of the c-myc locus. Karyotype analysis demonstrated that amplification of the c-myc locus was a result of duplication of the long arm of chromosome eight.
Blotting, Northern↗