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Biomedical subjects

M Y Hsu

Publications and source records attributed to M Y Hsu.

At least 19 recordsLinked to original sources
PubMed2001

Alpha5 and alpha2 integrin gene transfers mimic the PDGF-B-induced transformed phenotype of fibroblasts in human skin.

Platelet-derived growth factor (PDGF)-B is a proto-oncogene capable of transforming fibroblasts. Using adenoviral vectors, we tested whether endogenous PDGF-B expression in human skin xenotransplants leads to changes in the expression of alpha5 and alpha2 integrin subunits and whether integrin overexpression leads to PDGF-related changes in the skin. In vitro, transduction of fibroblasts with PDGF-B or the integrin alpha5 subunit stimulated multilayered growth and spindle-type morphology, both markers of mesenchymal cell transformation. In vivo, PDGF-B transduction of the human dermis was associated with up-regulation of collagen and fibronectin synthesis, increases in alpha5 and alpha2 integrin subunit expression, vessel formation, and proliferation of fibroblasts, keratinocytes, and pericytes. A similar stromal response was induced when alpha5 and alpha2 integrin subunits were overexpressed in the human dermis, suggesting that integrins play a major role in the induction of a transformed phenotype of fibroblasts by PDGF-B.

Antigens, CD↗
PubMed2000

Human melanoma progression in skin reconstructs : biological significance of bFGF.

Human skin reconstructs are three-dimensional in vitro models consisting of epidermal keratinocytes plated onto fibroblast-contracted collagen gels. Cells in skin reconstructs more closely recapitulate the in situ phenotype than do cells in monolayer culture. Normal melanocytes in skin reconstructs remained singly distributed at the basement membrane which separated the epidermis from the dermis. Cell lines derived from biologically early primary melanomas of the radial growth phase proliferated in the epidermis and the basement membrane was left intact. Growth and migration of the radial growth phase melanoma cells in the dermal reconstruct and tumorigenicity in vivo were only observed when cells were transduced with the basic fibroblast growth factor gene, a major autocrine growth stimulator for melanomas. Primary melanoma cell lines representing the more advanced stage vertical growth phase invaded the dermis in reconstructs and only an irregular basement membrane was formed. Metastatic melanoma cells rapidly proliferated and aggressively invaded deep into the dermis, with each cell line showing typical invasion and growth characteristics. Our results demonstrate that the growth patterns of melanoma cells in skin reconstructs closely correspond to those in situ and that basic fibroblast growth factor is critical for progression.

Basement Membrane↗
PubMed2000

E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors.

In human epidermis, functional symbiosis requires homeostatic balance between keratinocytes and melanocytes. Compelling evidence from co-culture studies demonstrated a sophisticated, multileveled regulation of normal melanocytic phenotype orchestrated by undifferentiated, basal-type keratinocytes. Keratinocytes control cell growth and dendricity, as well as expression of melanoma-associated cell surface molecules of normal melanocytes. In contrast, melanoma cells are refractory to the keratinocyte-mediated regulation. The loss of regulatory dominance by keratinocytes occurs in concert with down-regulation of E-cadherin expression in melanoma cells. To investigate the potential role of E-cadherin in melanoma-keratinocyte interaction, we transduced E-cadherin-negative melanoma cells with full-length E-cadherin cDNA using an adenoviral vector. Our results show that functional E-cadherin expression in melanoma cells leads to cell adhesion to keratinocytes rendering them susceptible for keratinocyte-mediated control. In a skin reconstruction model, ectopic E-cadherin expression inhibits invasion of melanoma cells into dermis by down-regulating invasion-related adhesion receptors, MelCAM/MUC18 and beta3 integrin subunit, and by induction of apoptosis. Thus, disruption of the E-cadherin-mediated, normal regulatory control from keratinocytes may represent one of the mechanisms accounting for melanocyte transformation.

Adenoviridae↗
PubMed2000

Determination of cyclamate in low-calorie foods by high-performance liquid chromatography with indirect visible photometry.

A rapid and simple method using reversed-phase high-performance liquid chromatography combined with indirect visible photometry at 433 nm was developed to determine cyclamate in some food samples. Cyclamate was not detected in these chosen samples as its use is banned in Hong Kong. Cyclamate can easily be detected in spiked samples using a mobile phase consisting of 30 mumol dm-3 Methyl Red and 0.02 mol dm-3 phosphate buffer (pH 7.0)-methanol in a volume ratio of 3:2. The column temperature was set at 23 degrees C. The detection limit was 0.14 mmol dm-3 and the relative standard deviation of the peak area response was 0.58% for a solution containing 5.0 mmol dm-3 of cyclamate (n = 8). This method was successfully applied to the analysis of eight spiked food samples and the cyclamate recoveries for these samples ranged from 93 to 99%.

Carcinogens↗
PubMed2000

A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium.

Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.

Amino Acid Sequence↗
PubMed1999

Basic fibroblast growth factor induces a transformed phenotype in normal human melanocytes.

Basic fibroblast growth factor (bFGF or FGF-2) is produced by nearly all melanomas in vitro and in vivo but not by normal melanocytes, which require exogenous bFGF for growth. In this study, we transduced normal human melanocytes to overexpress two forms of bFGF: (bFGF-Long and bFGF-Short) using replication-deficient adenovirus 5 vectors. bFGF-Long induced the 17.8, 22.5, 23.1 and 24.2 kDa forms of bFGF, whereas bFGF-Short induced only the 17.8 kDa mature form. Growth of cultured melanocytes transduced with either vector was similar to that of nevus and melanoma cells and was independent of exogenous bFGF and of insulin/insulin-like growth factor 1, and cyclic AMP enhancers, requiring only phorbol ester as an exogenous mitogen. Like primary melanoma cells, transduced normal melanocytes grew anchorage independently in soft agar. When injected into the dermis of human skin grafted to mice, bFGF-transduced melanocytes proliferated for at least 20 days, whereas cells from control cultures showed poor survival and no proliferation. These results demonstrate that bFGF upregulation is a critical component in melanoma progression.

Adenoviridae↗
PubMed1999

Highly specific recognition of primer RNA structures for 2'-OH priming reaction by bacterial reverse transcriptases.

A minor population of Escherichia coli contains retro-elements called retrons, which encode reverse transcriptases (RT) to synthesize peculiar satellite DNAs called multicopy single-stranded DNA (msDNA). These RTs recognize specific RNA structures in their individual primer-template RNAs to initiate cDNA synthesis from the 2'-OH group of a specific internal G residue (branching G residue). The resulting products (msDNA) consist of RNA and single-stranded DNA, sharing hardly any sequence homology. Here, we investigated how RT-Ec86 recognizes the specific RNA structure in its primer-template RNA. On the basis of structural comparison with HIV-1 RT, domain exchanges were carried out between two E. coli RTs, RT-Ec86 and RT-Ec73. RT-Ec86 (320 residues) and RT-Ec73 (316 residues) share only 71 identical residues (22%). From the analysis of 10 such constructs, the C-terminal 91-residue sequence of RT-Ec86 was found to be essential for the recognition of the unique stem-loop structure and the branching G residue in the primer-template RNA for retron-Ec86. Using the SELEX (systematic evolution of ligands by exponential enrichment) method with RT-Ec86 and primer RNAs containing random sequences, the identical stem-loop structure (including the 3-U loop) to that found in the retron-Ec86 primer-template RNA was enriched. In addition, the highly conserved 4-base sequence (UAGC), including the branching G residue, was also enriched. These results indicate that the highly diverse C-terminal region recognizes specific stem-loop structures and the branching G residue located upstream of the stem-loop structure. The present results with seemingly primitive RNA-dependent DNA polymerases provide insight into the mechanisms for specific protein RNA recognition.

Amino Acid Sequence↗
PubMed1999

Up-regulation of ephrin-A1 during melanoma progression.

Ephrin-A1, formerly called B61, is a new melanoma growth factor; it is angiogenic and chemoattractant for endothelial cells. EPH-A2, or ECK (a receptor for ephrin-A1), is ectopically expressed in most melanoma cell lines; the pathology where this expression is first manifested and the possible role of the receptor in tumor progression are unknown. To determine these, we studied the expression of this ligand and receptor in biopsies of benign and malignant melanocytic lesions. EPH-A2 was not detected in normal melanocytes, benign compound nevi or advanced melanomas, though it was found in 2 of 9 biopsies of malignant melanoma in situ. Ephrin-A1 was present in occasional early lesions and in advanced primary melanomas (43%) and metastatic melanomas (67%). Expression of ephrin-A1 was induced in melanoma cells by pro-inflammatory cytokines. Our findings are consistent with 2 possible roles for ephrin-A1 in melanoma development: it may promote melanocytic cell growth or survival and induce vascularization in advanced melanomas. Both effects may be potentiated by inflammatory responses. Our data are consistent with earlier observations that an inflammatory infiltrate is associated with poor prognosis in thin primary melanomas.

Cytokines↗
PubMed1999

Human xenografts, human skin and skin reconstructs for studies in melanoma development and progression.

Melanoma develops from a series of architectural and phenotypically distinct stages and becomes progressively aggressive. Considerable progress has been made in understanding the biological, pathological, and immunological aspects of human melanoma. Genetic and cytogenetic studies have revealed broad chromosomal abnormalities and wide mutational spectra. Precise biological and molecular determinants responsible for melanoma progression are not yet known. This is in part due to lack of experimental models that mimic human melanomas. Experimental models in melanoma should not only identify cause and origin of malignancy, but also should represent the ordered progression steps that culminate in metastasis to distant organs. Currently, there are several mouse and other vertebrate melanoma models under investigation; several of them promise to shed light on mechanisms of melanomagenesis. However, many of them suffer from lack of context to human skin architecture and hence, are of basic interest. The lack of appropriate models impeded the efforts to understand origin, etiology, progression and ultimately therapeutic benefits to humans. Development of human skin-mouse chimeric models has appeal because it mimics human diseases. In addition, human artificial skin constructs in vitro promises to be a versatile and efficient model to study not only origin and mechanisms of melanoma, but also progression. This review will focus on the recent progress in establishing tumor models in melanoma in general and their relevance to human melanoma as molecular determinants of tumor progression.

Animals↗
PubMed1998

Molecular events in melanoma development and progression.

Based on clinical and histopathological features, five steps of melanoma progression have been proposed: common acquired and congenital nevi with structurally normal melanocytes, dysplastic nevus with structural and architectural atypia, early radial growth phase (RGP) primary melanoma, advanced vertical growth phase primary melanoma (VGP) with competence for metastasis, and metastatic melanoma. Despite a wealth of research resources (tissues, cell lines, and antibodies), the genetic alterations responsible for the development and stepwise progression of melanoma are still unclear. Cytogenetic analyses have failed to identify consistent gene deletions, mutations, translocations, or amplifications in sporadic cases. However, in vitro characterization of melanoma cells has revealed fundamental differences from normal melanocytes. Earlier work using monoclonal antibodies has defined a variety of melanoma-associated antigens that mediate cell-cell or cell-substratum adhesion, growth regulation, proteolysis, and modulation of immune responses. Functional studies of these individual candidate molecules will lead to a better understanding of the pathogenesis of melanoma and of potential targets for rational therapy.

Antigens, Neoplasm↗
PubMed1998

Adenoviral gene transfer of beta3 integrin subunit induces conversion from radial to vertical growth phase in primary human melanoma.

Expression of the beta3 subunit of the alphavbeta3 vitronectin receptor on melanoma cells is associated with tumor thickness and the ability to invade and metastasize. To address the role of alphavbeta3 in the complex process of progression from the nontumorigenic radial to the tumorigenic vertical growth phase of primary melanoma, we examined the biological consequences of overexpressing alphavbeta3 in early-stage melanoma cells using an adenoviral vector for gene transfer. Overexpression of functional alphavbeta3 in radial growth phase primary melanoma cells 1) promotes both anchorage-dependent and -independent growth, 2) initiates invasive growth from the epidermis into the dermis in three-dimensional skin reconstructs, 3) prevents apoptosis of invading cells, and 4) increases tumor growth in vivo. Thus, alphavbeta3 serves diverse biological functions during the progression from the nontumorigenic radial growth phase to the tumorigenic and-invasive vertical growth phase primary melanoma.

Adenoviridae↗
PubMed1997

Melanoma cell-cell interactions are mediated through heterophilic Mel-CAM/ligand adhesion.

Mel-cell adhesion molecule (CAM), also known as MUC18 and CD146, is a novel member of the immunoglobulin supergene family. Mel-CAM was first identified as an integral membrane glycoprotein in human melanoma and is also abundantly expressed by endothelial cells of various origins. In a previous study (I. M. Shih et al., Cancer Res., 54: 2514-2520, 1994), we showed that Mel-CAM is a cell-cell adhesion molecule with a possible role in melanoma invasion and metastasis. Here, we define the molecular mechanism responsible for cell-cell adhesion of Mel-CAM and demonstrate its role in melanoma-endothelial cell interactions. Most of human melanoma cells, including Mel-CAM-negative SBcl-2 cells, adhered to nitrocellulose-immobilized Mel-CAM produced by baculovirus recombinants. This adhesion can be blocked by full-length Mel-CAM or polyclonal antiserum against Mel-CAM. Adhesion is not affected by the presence of EDTA, truncated Mel-CAM extracellular domain, or heparan sulfate proteoglycan. In cell aggregation assays, Mel-CAM-negative SBcl-2 cells cluster with U937TM cells (U937 transfected with Mel-CAM cDNA) but not with control nontransfectants, suggesting that SBcl-2 cells express the ligand for Mel-CAM. SBcl-2 cells also form heterotypic aggregates with Mel-CAM-positive human endothelial cells but not with Mel-CAM-negative but ligand-positive smooth muscle cells. Taken together, our results show that Mel-CAM mediates cell-cell adhesion through heterophilic adhesion to an as yet unidentified ligand present on melanoma but not on endothelial cells. Thus, melanoma-endothelial interactions during metastasis may occur through this novel mechanism.

Antigens, CD↗
PubMed1997

The cell-cell adhesion receptor Mel-CAM acts as a tumor suppressor in breast carcinoma.

Mel-CAM (MUC18 or CD146) is a cell adhesion molecule sharing sequence homology with members of the immunoglobulin gene superfamily. Mel-CAM was originally described as a marker associated with invasion and metastasis in melanoma. We determined here the distribution and biological significance of Mel-CAM in normal, benign proliferative, and neoplastic breast ductal epithelium. Using a Mel-CAM-specific monoclonal antibody, we, immunohistochemically demonstrate Mel-CAM expression in 14 of 14 (100%) normal breast epithelia and benign proliferative ductal epithelial lesions, whereas Mel-CAM expression can only be focally detected in 12 of 72 (17%) breast carcinomas. Solid-phase cell adhesion assay revealed that breast carcinoma cells in culture express the ligand for Mel-CAM. Transfection of Mel-CAM cDNA into breast carcinoma cells induces a more cohesive cell growth pattern and establishes smaller tumors in immunocompromised mice than mock transfectants. In conclusion, Mel-CAM is distributed throughout normal and benign proliferative mammary ductal epithelium, but it is frequently lost in carcinomas; it functions as a heterophilic cell-cell adhesion molecule in breast epithelium, and loss of Mel-CAM expression in breast carcinoma may be an important step for tumor progression.

Animals↗
PubMed1996

Shifts in cadherin profiles between human normal melanocytes and melanomas.

Direct contacts between keratinocytes and melanocytes play an important role in conserving the characteristic phenotype and biologic behavior of melanocytic cells. Although the mechanisms involved remain unclear, given the role of adhesion molecules in controlling cellular interactions, disturbances in normal keratinocyte-melanocyte adhesion mediated by cadherin may contribute to malignant transformation by releasing melanocytes from a variety of contact-mediated regulatory controls. To determine the potential clinical relevance of cadherin profiles in melanomas and to study their possible involvement in the phenotypic plasticity of melanocytic cells, we used immunostaining, biochemical, and co-culture techniques. Double immunofluorescence demonstrated expression of cadherins and their associating proteins, alpha- and beta-catenin, in melanocytes in situ. Melanomas were heterogeneous when evaluated immunohistochemically, with positive rates of four of 14, eight of 12, and 12 of 16 to anti-E-, anti-P-, and anti-N-cadherin monoclonal antibodies, respectively. Flow cytometry indicated abundant expression of E-cadherin but marginal P- and N-cadherin in cultured melanocytes. In contrast, only one (WM1232) of 16 melanoma cell lines tested was positive for E-cadherin, none was positive for P-cadherin, and all but one were positive for N-cadherin. Western blot confirmed E-cadherin expression in melanocytic cells. Immunoprecipitation further revealed complexes of E-cadherin with catenins in WM1232 melanoma cells. Co-culture studies indicated that only melanoma cells expressing E-cadherin (WM1232) were susceptible to keratinocyte-mediated control of the expression of the melanoma cell adhesion molecule, Mel-CAM. The results suggest downregulation of E-cadherin but upregulation of N-cadherin in melanoma cells. Such a shift in cadherin profiles may endow melanocytic cells with new adhesive properties and altered spatial relations that favor uncontrolled proliferation, migration, and invasion.

Antigens, Neoplasm↗
PubMed1995

Two recA genes in Myxococcus xanthus.

Two recA genes, recA1 and recA2, in Myxococcus xanthus were cloned by using the recA gene of Escherichia coli, and their DNA sequences were determined. On the basis of deduced amino acid sequences, RecA1 and RecA2 have 67.0% identity to each other and 60.5 and 60.9% identities to E. coli RecA, respectively. Expression of recA2 was detected in both vegetative and developmental cells by Northern blot (RNA) analysis, and a threefold induction was observed when cells were treated with nalidixic acid. Repeated attempts to isolate a recA2 disruption mutant have failed, while a recA1 disruption mutant was readily isolated. Both the recA1 and recA2 genes expressed in E. coli complement the UV sensitivity of an E. coli recA strain.

Amino Acid Sequence↗
PubMed1994

Regulation of Mel-CAM/MUC18 expression on melanocytes of different stages of tumor progression by normal keratinocytes.

The cell-cell adhesion receptor, Mel-CAM/MUC18, is highly expressed on metastatic melanoma cells and is also detectable on primary melanomas but not on normal melanocytes. Previous studies have shown that increased Mel-CAM/MUC18 expression correlates with tumor thickness and metastatic potential. We show here that normal melanocytes and nevus cells in culture express Mel-CAM/MUC18, but expression is down-regulated when cells are co-cultured with keratinocytes. Such keratinocyte-mediated regulation of Mel-CAM/MUC18 expression on melanocytes, nevus cells, and early melanomas can also be demonstrated in situ in patients' specimens. On the other hand, melanoma cells from primary and metastatic lesions constitutively express Mel-CAM/MUC18, and keratinocytes have no modulatory effect. These results suggest that contact between keratinocytes and human melanocytic cells modulates Mel-CAM/MUC18 expression, raising the possibility that escape from keratinocyte control during melanoma development leads to expression of antigens that contribute to the malignant phenotype.

Antigens, CD↗
PubMed1993

Reverse transcriptases from bacterial retrons require specific secondary structures at the 5'-end of the template for the cDNA priming reaction.

Multicopy single-stranded DNA (msDNA) is a peculiar molecule consisting of a single-stranded DNA that is branched out from an internal G residue of an RNA molecule (msdRNA) via a 2',5'-phosphodiester linkage. The genetic unit required for msDNA synthesis is designated "retron" and consists of msr (a gene for msdRNA), msd (a gene for msDNA), and a gene for reverse transcriptase (RT) in a single operon. To date, four different msDNAs have been isolated from Escherichia coli. They do not share any primary sequences in either RNA or DNA. To elucidate the specificity of bacterial RT for msDNA synthesis, the msr-msd region from retron-Ec67 was introduced into E. coli cells producing RT-Ec73, or the msr-msd region from retron-Ec73 into E. coli cells producing RT-Ec67. In both cases, msDNA was not synthesized. However, when the msdRNA coding regions (msr) for retron-Ec67 and -Ec73 were mutually exchanged and the chimeric genes were introduced into E. coli cells producing either RT-Ec67 or RT-Ec73, it was thus found that msDNA was produced only when msr and RT were from the same retron. Requirement of the msr region for msDNA synthesis by RT was further investigated by mutations in the msr region for retron-Ec67. These analyses revealed that there is a strict requirement for specific primary sequences as well as the secondary structure in msdRNA. This finding is discussed in relationship to the mechanism of the priming reaction of cDNA synthesis by eukaryotic retroviral RTs using tRNAs.

Bacterial Proteins↗