Search PubMed⌕ Search

Biomedical subjects

L Sherman

Publications and source records attributed to L Sherman.

At least 37 records · Page 2Linked to original sources
PubMed1999

Increased expression of the NF2 tumor suppressor gene product, merlin, impairs cell motility, adhesionand spreading.

The neurofibromatosis 2 ( NF2 ) gene product, merlin, is a tumor suppressor protein mutated in schwanno-mas and several other tumors. Merlin, which shares significant homology with the actin-associated proteins ezrin, radixin and moesin (ERM proteins), inhibits cell growth when overexpressed in cell lines. The similarities between merlin and ERM proteins suggest that merlin's growth-regulatory capabilities may be due to alterations in cytoskeletal function. We examined this possibility in rat schwannoma cell lines overexpressing wild-type merlin isoforms and mutant merlin proteins. We found that overexpression of wild-type merlin resulted in transient alterations in F-actin organization, cell spreading and cell attachment. Merlin overexpression also impaired cell motility as measured in an in vitro motility assay. These effects were only observed in cells overexpressing a merlin isoform capable of inhibiting cell growth and not with mutant merlin molecules (NF2 patient mutations) or a merlin splice variant (isoform II) lacking growth-inhibitory activity. These data indicate that merlin may function to maintain normal cytoskeletal organization, and suggest that merlin's influence on cell growth depends on specific cytoskeletal rearrangements.

Animals↗
PubMed1999

The E2 protein of human papillomavirus type 16 is translated from a variety of differentially spliced polycistronic mRNAs.

The major regulation protein of human papillomavirus (HPV) transcription is the viral E2 protein. Previous studies have identified a variety of alternatively spliced mRNAs containing multiple open reading frames (ORFs) encoding the E2 protein of HPV type 16. In these mRNAs the E2 ORF is contained as an internal ORF. In the present study, the translational capacities of three mRNA species starting at the p97 promoter and containing the 880/2581, 880/2708 and 226/2708 splice junctions upstream of the E2 ORF were investigated. Partial cDNAs spanning the E2 ORF and the related upstream ORFs were synthesized and assessed for E2 protein translation in vivo, in COS cells, and in vitro, in cell-free systems. Results of these analyses indicated that E2 protein was translated from all three mRNAs. Translation efficiency of E2 from the natural polycistronic templates was lower compared with that from a synthetic monocistronic control. Translation from the d-type bicistronic template (226/2708) was more efficient than that from the a-type (880/2708) and a'-type (880/2561) polycistronic templates. Further investigation of the translation of proteins encoded by the ORFs preceding the E2 ORF showed that a- and a'-type templates served for translation mainly of E7 but also of E61, while the d-type template served for translation of E6IV. Overall, the translation data support the suggestion that the corresponding mRNAs may function as polycistronic transcripts.

Alternative Splicing↗
PubMed1998

Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy.

Replication-defective adenoviral (RDAd) vectors can be generated at high titers and infect both dividing and nondividing cells. Long term expression in the transduced tissue, however, has been a problem because of the cellular immune responses against the infected cells. We demonstrate that mice injected with RDAd vectors containing mouse leptin gene reduce food intake and lose weight for only 7 to 10 days. Splenocytes obtained from infected mice are able to lyse target cells infected with RDAd vectors. Surprisingly, target cells infected with psoralen-treated, UV-crosslinked, biologically inactive RDAd also were lysed efficiently by the effector cells. Furthermore, splenocytes obtained from mice injected with inactive RDAd vectors efficiently lysed target cells infected with RDAd vectors. Whether RDAd vectors were injected i.m. or i.v. or through an i.p. route, the extent of lysis was similar. We propose that cells infected with RDAd vectors present antigens for recognition by class 1 major histocompatibility complex-restricted cytotoxic T lymphocytes by a mechanism that does not require viral replication or de novo protein synthesis. These results should prompt reevaluation of the use of RDAd vectors for gene therapy when long-term expression is required.

Adenoviridae↗
PubMed1998

A splice variant of CD44 expressed in the apical ectodermal ridge presents fibroblast growth factors to limb mesenchyme and is required for limb outgrowth.

Signals from the apical ectodermal ridge (AER) of the developing vertebrate limb, including fibroblast growth factor-8 (FGF-8), can maintain limb mesenchymal cells in a proliferative state. We report here that a specific CD44 splice variant is crucial for the proliferation of these mesenchymal cells. Epitopes carried by this variant colocalize temporally and spatially with FGF-8 in the AER throughout early limb development. A splice variant containing the same sequences expressed on model cells binds both FGF-4 and FGF-8 and stimulates mesenchymal cells in vitro. When applied to the AER, an antibody against a specific CD44 epitope blocks FGF presentation and inhibits limb outgrowth. Therefore, CD44 is necessary for limb development and functions in a novel growth factor presentation mechanism likely relevant in other physiological and pathological situations where a cell surface protein presents a signaling molecule to a neighboring cell.

Alternative Splicing↗
PubMed1998

Induction of apoptosis in human keratinocytes containing mutated p53 alleles and its inhibition by both the E6 and E7 oncoproteins.

Induction of apoptosis is a function of external stimuli and cellular gene expression. Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled by elevation of p53 levels. We have investigated the response of immortalized human keratinocytes (HaCaT) bearing mutated alleles of p53 to genotoxic stress and the effect of human papillomavirus (HPV) 16 E6 and E7 on this response. UVC irradiation triggered HaCaT's cell death with several characteristics of apoptosis, including DNA laddering, chromatin condensation and fragmentation, and the appearance of cells with a low content of DNA (categorized as sub-G1 by cell sorter analysis). This response was accompanied by accumulation of cells in S phase of the cell cycle. HaCaT cells infected with retroviruses carrying HPV16 E6 or E7 showed a significant reduction in their apoptotic response, which was not observed in cells infected with the LXSN vector DNA-carrying virus. Reduced apoptosis in HaCaT cells expressing E6 or E7 also was observed after treatment with the alkylating agent mitomycin C. Western blot analysis of p53 and p21/WAF-1/CIP-1, a downstream effector of p53, did not reveal any changes in the levels of these proteins after UVC irradiation in either HaCaT cells or HaCaT cells expressing HPV16 E6 or E7.

Alleles↗
PubMed1998

HPV-16 E7 protein bypasses keratinocyte growth inhibition by serum and calcium.

The E6 and E7 genes of HPV-16 or HPV-18 both are necessary for effective immortalization of primary human genital keratinocytes. To analyse the individual role of E6 and E7 genes in dysregulating cell growth, we cloned the HPV-16 E6, E7 and E6/E7 genes into retroviruses. Primary human keratinocytes (PHK) were then infected with these retroviruses and selected in differentiation-inducing medium (high calcium and serum). The E6/E7 retroviruses were the most effective at inducing differentiation-resistant colonies. Intermediate numbers of colonies were induced by E6 and low numbers by E7. Interestingly, only cultures infected with E7 and E6/E7 retroviruses showed a significant proportion of cells progressing into the S phase, consistent with our earlier studies showing that E7 is required for the efficient immortalization of genital keratinocytes. Accompanying this entry into S phase, the E7 or E6/E7 transduced cells expressed high levels of cyclins A, B and E, but lower levels of cyclin D. In addition, cdc-2, cdk-2 and cdk-4 were also increased. No significant differences were detected in the expression of c-myc and c-fos between the vector and any of the transduced cells. Keratinocytes infected with the E7 retrovirus exhibited decreased levels of Rb protein and increased levels of p53, whereas cells infected with E6-expressing retroviruses displayed normal levels of Rb protein and decreased levels of p53. Finally, E7 induced a three-fold increase in bcl-2 expression. Our results indicate that the HPV-16 E7 gene alone is sufficient to bypass keratinoctye growth arrest induced by serum and calcium exposure and that the discordant expression of several cell regulatory proteins accompanies this unregulated proliferation.

Calcium↗
PubMed1998

How tumor cells make use of CD44.

A variant of CD44 containing exon v3 sequences is expressed in the apical ectodermal ridge of the limb bud during embryogenesis. This variant is modified by heparan sulfate moieties and acts as low affinity receptor for FGFs. These FGFs are presented by CD44 to mesenchymal cells which induces their proliferation and limb outgrowth. We suggest that a similar growthfactor presentation mechanism accounts for the function of CD44 variants on metastasizing tumor cells.

Animals↗
PubMed1997

Interdomain binding mediates tumor growth suppression by the NF2 gene product.

The neurofibromatosis 2 (NF2) tumor suppressor gene encodes an intracellular membrane-associated protein, called merlin (or schwannomin), that belongs to the band 4.1 family of cytoskeleton-associated proteins. Inactivating NF2 mutations occur in several sporadic tumor types and have been linked to the NF2 disease, whose hallmark is the development of bilateral Schwann cell tumors (schwannomas) of the eighth cranial nerve. Two major alternatively spliced NF2 variants are expressed in normal tissues: 'NF2-17' lacking exon 16 and 'NF2-16' that contains exon 16 and encodes a merlin protein truncated at the C-terminus. We report that overexpression of NF2-17 in rat schwannoma cells inhibits their growth in vitro and in vivo, while NF2-16 fails to influence schwannoma growth. Tumor growth inhibition by merlin depends on an interdomain association occurring either in cis or in trans between the N- and C-termini. This association does not occur in the truncated NF2-16 protein nor in a mutant NF2-17 protein lacking C-terminal sequences. These data indicate that merlin has a unique mechanism of tumor suppression, inhibiting cell proliferation via self-association.

Animals↗
PubMed1997

CD44 expression is aberrant in benign Schwann cell tumors possessing mutations in the neurofibromatosis type 2, but not type 1, gene.

Atypical expression of CD44 splice variants has been implicated in the progression of numerous tumors. This abnormal CD44 expression is presumed to result from gene alterations that cause tumorigenic transformation. Two tumor types that have been linked to specific gene alterations are schwannomas, which have mutations in the neurofibromatosis (NF) type 2 (NF2) gene, and neurofibromas, which characteristically possess NF type 1 (NF1) gene mutations. We examined CD44 expression in normal sciatic nerves, in schwannomas with confirmed NF2 mutations, and in neurofibromas and malignant peripheral nerve sheath tumor tissue and cell lines from NF1 patients. Compared to normal nerves, schwannomas express higher total levels of CD44 and additional splice variants, whereas CD44 expression in neurofibromas is unaltered. Malignant peripheral nerve sheath tumor tissue and cell lines express the CD44v6 epitope, which is not expressed by normal Schwann cells or by other Schwann cell tumors. These data indicate that altered CD44 expression correlates strictly with mutations in the NF2 but not NF1 gene and suggest that CD44v6 might be a marker for the malignant transformation of Schwann cells.

Alternative Splicing↗
PubMed1997

Inhibition of serum- and calcium-induced differentiation of human keratinocytes by HPV16 E6 oncoprotein: role of p53 inactivation.

We have recently shown that human papillomavirus (HPV16) E6 oncoprotein exhibits two separate biological activities in genital keratinocytes (PHKs). E6 protein by itself is capable of inducing colonies of proliferating cells resistant to serum and calcium-induced differentiation, whereas both E6 and E7 are required for immortalization of PHK. Using epitope-tagged E6 carboxy-terminal truncation mutants, we mapped the domain between amino acid residues 132 and 141 as being essential for the induction of differentiation resistance (L. Sherman and R. Schlegel, J. Virol. 70, 3269-3279, 1996). To determine whether E6 protein's ability to alter PHK response to serum and calcium was associated with its ability to inactivate p53, we evaluated each of the above E6 mutants and three E6 natural variants in these respective assays. Our results demonstrate that the E6 region spanning residues 132-141 is required for p53 degradation and for abrogation of p53 transactivation, suggesting a possible correlation between E6 biological activity in altering differentiation and loss of p53 function. To evaluate whether selective inactivation of p53 is sufficient for altering the response of PHK to serum and calcium we investigated the capacity of plasmids encoding a dominant mutant human p53 and human MDM-2 to functionally substitute for E6 in colony formation in PHK. Plasmids were verified for their ability to inactivate wild-type p53 by testing their capacity to abrogate the p53 transactivation function. The results obtained showed that vectors encoding human MDM-2 and mutant p53, while active in inhibition of p53-dependent transactivation and capable of expressing stable proteins in PHK, failed to induce colonies of proliferating cells resistant to serum and calcium differentiation. These data argue that p53 inactivation may not be the sole E6 function required for altering the response of PHK to serum- and calcium-triggered differentiation.

Blood Proteins↗
PubMed1997

The human papillomavirus type 16 E6 and E7 oncoproteins dissociate cellular telomerase activity from the maintenance of telomere length.

The "high risk" subgroup of human papillomaviruses (e.g. HPV-16 and HPV-18) infect and induce tumors of mucosal epithelium. These neoplasms, which can progress to malignancy, retain and express the papillomavirus E6 and E7 oncogenes. In vitro, the E6 and E7 proteins associate with the cellular p53 and Rb proteins and interfere with their normal growth-regulatory functions. We report here that primary human keratinocytes transduced with the HPV-16 E6 gene, but not the E7 gene, express significant telomerase activity. However, despite this detectable enzymatic activity, E6-transduced cells continue to shorten their telomeres during in vitro passaging similar to control cells and to cells expressing the E7 and E6+E7 genes. At late passages, however, E7-transduced cells partially restore telomere length, although they lack detectable telomerase activity, demonstrating that E6-independent, telomerase-independent events mediate this change.

Cell Line↗
PubMed1996

The E6 variant proteins E6I-E6IV of human papillomavirus 16: expression in cell free systems and bacteria and study of their interaction with p53.

Several species of alternatively spliced mRNAs are transcribed from the E6 gene region of human papillomavirus (HPV) 16. These have the coding capacity for either the full length E6 of 151 amino acids (aa) or four truncated variants, E6I-E6IV, of 43-64 aa. As the first step to identify the putative E6 variants and their functions, we generated cDNAs corresponding to the various E6 open reading frames (ORF) and examined their expression employing in vitro transcription/translation systems and the bacterial pET system. In wheat germ extract, in vitro translation resulted in the production of all five proteins, E6 and E6I-E6IV. These proteins were also expressed as stable fusion proteins from the pET16b and pET17 x b vectors in Escherichia coli. Mobilites of the E6 variant proteins on SDS-acrylamide gels were consistent with their predicted sizes. The authenticity of the synthesized proteins was confirmed by immunoprecipitation with specific antibodies directed against epitopes in the N-terminal portion of E6 as well as antibodies raised against the individual variant proteins produced in E. coli. In rabbit reticulocyte lysate, however, only the full length E6 and the E6IV variant were synthesized. This could be due to inefficient translation as well as lower stability of the short variants. E6I-III, in reticulocyte lysate (RTL). The ability of the E6 variants to associate with p53 and target its proteolytic degradation in vitro, was examined in coimmunoprecipitation assays, using in vitro synthesized proteins and monoclonal antibodies to p53. Results of these assays indicated that only the full length E6 efficiently binds to and promotes the degradation of p53. The E6 variants E6I-E6IV, although able to associate with p53 at a low efficiency, were unable to target its degradation.

Amino Acid Sequence↗
PubMed1996

Earth benefits of interdisciplinary CELSS-related research by the NSCORT in Bioregenerative Life Support.

Earth benefits of research from the NSCORT in Bioregenerative Life Support will include the following: development of active control mechanisms for light, CO2, and temperature to maximize photosynthesis of crop plants during important phases of crop development; automation of crop culture systems; creation of novel culture systems for optimum productivity; creation of value-added crops with superior nutritional, yield, and waste-process characteristics; environmental control of food and toxicant composition of crops; new process technologies and novel food products for safe, nutritious, palatable vegetarian diets; creation of menus for healthful vegetarian diets with psychological acceptability; enzymatic procedures to degrade recalcitrant crop residues occurring in municipal waste; control-system strategies to ensure sustainabilty of a CELSS that will enable management of diverse complex systems on Earth.

Biomass↗