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L Banks

Publications and source records attributed to L Banks.

At least 73 records · Page 4Linked to original sources
PubMed1992

Human papillomavirus type 16 E5 gene stimulates the transforming activity of the epidermal growth factor receptor.

We have until recently made several unsuccessful attempts to assign any activity to the human papillomavirus type 16 (HPV-16) E5 gene product. However, studies with the bovine papilloma virus 1 (BPV-1) E5 protein indicated an interaction with the epidermal growth factor receptor (EGFR). In light of the overall similarity between the HPV and BPV E5 proteins we attempted to determine whether the HPV-16 E5 gene had any common activity. In cells expressing high levels of EGFR plus HPV-16 E5 we found a dramatically increased proliferative activity in soft-agar assays in the presence of EGF. The specificity of this activity was monitored by the addition of other mitogenic agents. The phorbol ester phorbol 12-myristate 13-acetate (PMA) had no effect on the E5-containing cells, although insulin weakly stimulated their growth in soft agar. Further analysis revealed the same number of EGF receptors were present on the E5-containing cells as on the control cells, although the E5 cells were more sensitive to lower concentrations of EGF. These results imply that E5 is amplifying the mitogenic signals from the EGFR in an as yet unknown manner, but which may form the basis of interactions with a variety of growth factor receptors. This report brings to three the number of transforming genes encoded by HPV-16.

3T3 Cells↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

Expression of the human papillomavirus E7 oncogene during cell transformation is sufficient to induce susceptibility to lysis by activated macrophages.

Human papillomaviruses (HPV), and in particular HPV type 16, are etiologic agents in the development of cervical cancer, which is the second most common form of cancer in women worldwide. Mammalian cells are susceptible to transformation in vitro by the E6 and E7 oncogenes derived from the HPV-16 genome. NIH-3T3 cells transfected with the HPV-16 E7 oncogene were found to exhibit cytolytic susceptibility to murine-activated macrophages. In comparison, E6 oncogene-expressing cells were not susceptible to lysis by activated macrophages. The E7 oncoprotein is multifunctional, being capable of complexing with the retinoblastoma tumor suppressor gene (anti-oncogene) product, stimulating DNA synthesis, and causing cell transformation in vitro. Macrophage killing assays performed on cell lines expressing E7 mutants revealed that the ability to complex the retinoblastoma tumor suppressor gene product and stimulate DNA synthesis did not induce susceptibility to activated macrophages, whereas the ability of E7 to cause transformation was required to induce susceptibility to activated macrophages. These data suggest that cell transformation is a more important prerequisite for inducing susceptibility to activated macrophages than is the loss of tumor suppressor gene function. This study also provides an initial link between HPV-16 oncogene expression and the ability of activated macrophages to selectively recognize and destroy HPV-16-associated neoplastic cells.

Amino Acid Sequence↗
PubMed1991

Loss of HPV-16 E7 dependence in cells transformed by HPV-16 E7 plus EJ-ras correlates with increased c-myc expression.

We have previously shown that continued expression of the HPV-16 E7 gene was required to maintain the transformed phenotype of primary baby rat kidney cells transformed by HPV-16 E7 plus EJ-ras. In this study we aimed to investigate the possible mechanisms by which cells could overcome this continued requirement for E7 expression. Using an inducible system for expression of the E7 gene we were able to generate cell lines no longer dependent on continued E7 expression. All such lines were still fully transformed, maintained inducible E7 expression and showed increased proliferative activity in the presence of HPV-16 E7 gene product. Further analysis has demonstrated a marked increase in the presence of c-myc protein in these cell lines when compared with the parental cells, suggesting a possible mechanism by which E7 dependence can be overcome.

Animals↗
PubMed1990

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↗
PubMed1990

Transformation of primary BRK cells by human papillomavirus type 16 and EJ-ras is increased by overexpression of the viral E2 protein.

The close association between human papillomavirus type 16 (HPV-16) and cervical cancer implies some role for the virus in the development of this disease. Recent studies have shown that HPV-16, under the control of strong heterologous promoters, can cooperate with the activated ras oncogene to transform primary baby rat kidney cells. Virus types associated with benign lesions, e.g. HPV-6 and -11, do not function in this system. The discrimination between virus types associated with benign and tumorigenic lesions by this assay implicate it as a useful system for the study of transformation in vitro. The studies reported here investigate the activity of the HPV-16 early gene product E2 in transformation. In the presence of exogenous E2, endogenous viral promoters are stimulated sufficiently to give a high efficiency of transformation in primary epithelial cells. This transactivation by E2 obviates the need for heterologous promoters, and implicates increased viral gene expression as a prerequisite for transformation. The stimulatory effect of E2 appears to be mediated through increased levels of expression of the E7 protein, which has been shown in similar assays to be sufficient to give transformation in cooperation with ras. CAT assays confirm that HPV-16 E2 can transactivate the HPV-16 early promoters. These studies demonstrate some of the elements in a complex series of events likely to be involved in the development of cervical carcinomas.

Animals↗
PubMed1990

Ability of the HPV16 E7 protein to bind RB and induce DNA synthesis is not sufficient for efficient transforming activity in NIH3T3 cells.

Previous studies have demonstrated that the HPV-16 E7 gene product encodes the major transforming activity of the virus in rodent cell systems. In this study we have generated a series of point mutations affecting the region of HPV-16 E7, which shows homology to adenovirus E1a conserved domain (CD)1. In conjunction with previously described mutants in the region of E7 with similarity to E1a CD2 and SV40 LT, we have investigated three known activities of the E7 protein; transformation, association with the cellular RB protein and induction of cellular DNA synthesis. The results show that RB binding correlates with the ability of E7 to induce cellular DNA synthesis and mediate cell transformation. In addition an unidentified function of E7, which is necessary for transformation of NIH3T3 cells, but does not affect RB binding or the ability to induce cellular DNA synthesis, has also been demonstrated. This study therefore identifies two separate regions of the E7 gene necessary for transformation of established cells. One of these, in the region of E7 which shows similarity to E1a CD2 and LT, is required for RB binding and DNA synthesis. The other region important for transformation, in the N-terminus of E7, is separable from the RB binding/DNA synthesis function.

Amino Acid Sequence↗
PubMed1990

HPV-16 E7 functions at the G1 to S phase transition in the cell cycle.

Previous reports have demonstrated that E7 is the major transforming gene of HPV-16 and that continued expression of the gene is required to maintain the transformed phenotype of primary baby rat kidney cells transformed by HPV-16 E7 and EJ-ras. To investigate the point of action of E7 in the cell cycle we have utilised a system of inducible expression of the E7 gene. The studies reported here show that stimulation of cellular DNA synthesis by E7 is distinct from that observed with calf serum. In combination with cytofluorimetric analyses these results indicate that E7 functions at the transition from G1 to S phase of the cell cycle. This adds further support to the hypothesis of a common pathway of transformation shared by the DNA tumour viruses HPV, SV40 and Adenovirus.

Animals↗
PubMed1989

Continued expression of HPV-16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV-16 plus EJ-ras.

The close association between HPV-16 and cervical cancer implies some role for the virus in development of this cancer. Recent studies have shown that the HPV-16 E7 gene encodes the major transforming activity of the virus in baby rat kidney (BRK) cell transformation assays. To investigate the requirement for continued E7 expression in BRK cells transformed by HPV-16 E7 plus EJ-ras, we have developed a system for inducible expression of the E7 gene. The studies reported here show that continued expression of the HPV-16 E7 gene is required for maintenance of the transformed phenotype in these cells. The implications these observations bear on the role of the E7 gene in cervical carcinoma are discussed.

Animals↗
PubMed1989

Amplification and overexpression of c-myc proto-oncogene correlate with the loss of glucocorticoid dependence in rodent cells transformed by human papillomavirus type 16.

Transformation of baby mouse kidney epithelial cells by human papillomavirus (HPV) type 16 is dependent both upon the cooperating oncogene and on the hormonal conditions after transfection. With v-fos as the oncogene, the transformed cells require glucocorticoid hormone, such as dexamethasone, for proliferation. This requirement is lost on continued passage of cell lines, and the cells become dexamethasone independent. Steroid-independent cell lines are also produced by growth of the HPV16/v-fos cells in 17 beta-estradiol following transfection, but cell lines produced in this manner showed no subsequent requirement for estradiol or dexamethasone. Expression of the c-myc proto-oncogene was measured in dexamethasone-dependent and independent cell lines. Dexamethasone-dependent cell lines all exhibited low level c-myc expression, but this markedly increased in the cell lines that had become dexamethasone independent as a result of continued in vitro growth. The low level of c-myc expression in some early passage dexamethasone-dependent cell lines appears to be associated with rearrangement of the c-myc locus, whereas late passage dexamethasone-independent cell lines contain amplified c-myc sequences. Dexamethasone-independent cell lines derived by growth in 17 beta-estradiol showed higher levels of c-myc expression, together with higher c-myc copy number, than dexamethasone-dependent lines. Taken together, these studies indicate that the steady-state level of c-myc expression affects the continued requirement of HPV16-transformed cells for dexamethasone.

Cell Division↗
PubMed1989

Lymphoproliferative response to fusion proteins of human papillomaviruses in patients with cervical intraepithelial neoplasia.

The cell-mediated immune response (CMI) to E6 and E4 fusion proteins of human papillomavirus type 16 (HPV-16), E6 fusion protein of HPV-18, and to control proteins similarly produced, was analysed in 29 patients with cervical intraepithelial neoplasia (CIN) and in 15 age-matched laboratory personnel using a lymphocyte proliferation assay (LPA). Compared to controls without any added proteins, a positive response (stimulation index greater than 2.0) to the highly purified E6 control protein was found in only one patient. Positive responses to the E4 control protein which contained beta-galactosidase were noted in three patients and two controls. With control proteins as baseline, the lymphocytes from nine patients (28%) and three laboratory personnel (20%) responded to at least on HPV fusion protein after 7 days in culture. Stimulation indices were low in both groups with a range of 2.06-4.69 and the difference in incidence of positive responses between the groups is not significant. Proliferative responses to HPV-1 and HPV-2 virion antigens were noted in 6/23 (26%) of the patients and 2/15 (18%) of the other group. No correlation between responsiveness and degree of dysplasia or presence of koilocytes was found in the patient group. The relevance of the low proliferative responses is discussed.

Adult↗
PubMed1989

Children's ability to make probability estimates: skills revealed through application of Anderson's functional measurement methodology.

Functional measurement methodology was used to assess children's attention to the total number of alternative outcomes as well as the number of target outcomes when making probability estimates. In Study 1, first-, third-, and fifth-grade children were given the task of estimating on a simple, continuous but nonnumeric scale the probability of drawing a particular color of jelly bean from a bag containing either 1, 2, or 3 jelly beans of that color, and either 6, 8, or 10 jelly beans total. In Study 2, first- through fifth-grade children were given the task of estimating the likelihood that a bug would fall on a pot containing a flower when presented displays of planters containing either 2, 3, 4, or 5 pots with flowers, and 6, 8, or 10 pots total. In both studies, the children were exposed to each of the combinations of numerator and denominator across 3 replications. The results indicate that all age groups attend to variations in the denominator as well as to variations in the numerator, and, furthermore, that they attend to the interaction between these variables. This finding contrasts sharply with research that requires children to choose which of 2 containers offers the greater chance of yielding a target item in a blind draw. It is suggested that children possess the skill to make accurate probability estimates, but they are unaware that these estimates should always be made and used when comparing the probability of an event across trials. The findings are discussed in relation to the broader issue of the limitations of the choice paradigm as a means of investigating children's thinking.

Attention↗
PubMed1988

Transformation of primary human fibroblast cells with human papillomavirus type 16 DNA and EJ-ras.

Human papillomavirus type 16 (HPV type 16) has been implicated as an etiological agent in human cervical cancer. This virus contains sequences which, under the proper transcriptional control, can increase the tumorigenicity of established mouse cells and cooperate with EJ-ras in transforming primary baby rat kidney (BRK) cells. These data argue that this virus contains oncogenic sequences. Because this is a human virus, it was important to study the effect on primary human cells of HPV type-16 DNA in both the presence and absence of EJ-ras. We now present data which demonstrate that HPV type-16 DNA, under the transcriptional control of Moloney murine leukemia viral long terminal repeats (MoMuLV-LTR), can extend the life span of primary human fibroblast cells in culture. Co-transfection of the HPV type-16 DNA containing plasmid together with an activated EJ-ras oncogene gives rise to transformed cells which grow faster, are morphologically different from and more aneuploid than cells established with only HPV type-16 DNA. Molecular analysis of these established and partially transformed human cells reveals that they contain and express the transfected DNA. These data argue that primary human cells can be transformed with HPV type-16 and EJ-ras sequences, whereas cells harboring only HPV type 16 DNA are largely normal but have an extended life span.

Animals↗
PubMed1988

Analysis of human papillomavirus sequences in cell lines recently derived from cervical cancers.

The DNA and RNA from four cell lines recently derived from cervical carcinomas (HX151c, HX155c, HX156c, and HX160c) were analyzed for the presence of human Papillomavirus DNA. Each contained HPV-16 DNA in a multicopy integrated form of varying complexity. Each also expressed RNA transcripts of similar sizes to CaSki cell transcripts. The splice acceptor position within the E6 coding region of the cell line HX156c was identical to CaSki, SiHa, and HeLa cells as well as a cell line derived from mouse fibroblasts transformed with HPV-16 sequences in a retrovirus vector. Immunoprecipitation with an anti-HPV-16 E6 polyclonal antiserum demonstrated that HX160c cells contain the E6 polypeptide derived from unspliced transcripts.

Base Sequence↗
PubMed1988

Comparison of the in vitro transforming activities of human papillomavirus types.

The association of certain human papillomavirus (HPV) types with the majority of human cervical carcinomas suggests a role for the virus in the development of this type of cancer. In this paper, we have examined the transforming properties of several HPV types where the early region genes of the virus are under the control of a strong heterologous promoter and show that major differences exist between the HPV types in their ability to transform primary rat kidney epithelial cells in conjunction with an activated ras oncogene. Those HPV types most commonly found in carcinomas--types 16, 18, 31 and 33--are capable of co-operating with ras to transform primary cells, but those types most commonly found in benign lesions--types 6 and 11--are not. We further demonstrate that the E7 gene of HPV16 by itself is sufficient to co-operate with activated ras to produce transformed cells which are tumorigenic in immunocompetent animals.

Adenocarcinoma↗
PubMed1988

Analysis of human papillomavirus type 16 polypeptides in transformed primary cells.

The close association between human papillomavirus type 16 and cervical cancer implies some role for the virus in the development of this cancer. It has been demonstrated that HPV-16 DNA sequences in the presence of activated ras are capable of transforming primary baby rat kidney cells. This communication describes the characterization of these transformed cells. All cell lines are shown to be tumorigenic in immunocompetent rats and there is continued expression of the HPV-16 E6 and E7 polypeptides in a number of the cell lines analyzed, suggesting a role for at least one of these proteins in the maintenance of the transformed phenotype.

Animals↗