Co-expression of epidermal growth factor-receptor and c-erb B-2 proto-oncogene product in human salivary-gland adenocarcinoma cell line HSG and the implications for HSG cell autocrine growth

Kyakumoto, Seiko; Kurokawa, Riki; Hoshino, Masayuki; M, Ota

doi:10.1016/0003-9969(94)90132-5

Cited by 7 publications

(7 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3 and 9). We confirmed that all mAbs specifically recognized HSP90 endogenously expressed in human salivary gland adenocarcinoma (22) and T47D cells, and we found that most of the mAbs cross-reacted with rat, mouse, and rabbit HSP90s.…”

Section: Discussionsupporting

confidence: 64%

Domain Structures and Immunogenic Regions of the 90-kDa Heat-shock Protein (HSP90)

Nemoto

Sato

Iwanari³

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

Domain structures of the 90-kDa heat-shock protein (HSP90) have been investigated with a library of anti-HSP90 monoclonal antibodies (mAbs) and by limited proteolysis with trypsin and chymotrypsin. Thirty-three mAbs were obtained by immunization with bacterially expressed human HSP90␣ and HSP90␤ isoforms. Among them, ten and three mAbs reacted specifically with HSP90␣ and HSP90␤, respectively. Immunoblotting and enzyme-linked immunosorbent analyses revealed that major immunogenic domains were located at two restricted regions of HSP90␣, i.e. amino acids 227-310 (designated Region I) and 702-716 (Region II), corresponding to a highly charged region and a region near the C terminus, respectively. Taken together with the characteristics of the amino acid sequences, these two immunogenic regions appeared to be exposed at the outer surface of HSP90. We further investigated the domain structures of HSP90 by limited proteolysis in combination with N-terminal sequencing and immunoblotting analyses. Tryptic cleavages of HSP90␣ at low concentrations revealed the existence of major susceptible sites at Arg 400 -Glu 401 , Lys 615 -Ala 616 , and Arg 620 -Asp 621 . Proteolysis at higher trypsin concentrations caused successive cleavages only toward the N-terminal direction from these sites, and Region I was included in the region selectively deleted during this process, thereby further suggesting its surface location. From these results, we propose three domain structures of HSP90 consisting of amino acids 1-400, 401-615, and 621-732. Differences in the protease sensitivity and immunogenicity further suggest that every domain is composed of two subdomains. This is the first study describing the domain structures and the immunogenic regions of HSP90.The 90-kDa heat-shock protein (HSP90) 1 is one of the major stress proteins in eukaryotic cells. There are at least two HSP90 genes, and two HSP90 isoform proteins, ␣ and ␤, are HSP90 is believed to have a chaperone-like activity for particular molecules that are involved in signal transduction, such as steroid receptors (6), casein kinase II (7), pp60 v-src (8), elF2␣ kinase (9), and aryl hydrocarbon receptor (dioxin receptor) (10). HSP90 specifically binds to these proteins; and, in most cases, this interaction is essential for the function of the proteins (9,11,12). However, a variety of evidence, i.e. the abundance of HSP90 in cells even under nonstressed conditions, the conserved amino acid sequences from prokaryotic to eukaryotic cells (13), and the indispensability in yeast (3), strongly suggests that HSP90 is involved in more fundamental functions of cells. In fact, several studies have recently shown that HSP90 functions as a general chaperone. That is, it interacts with various proteins less specifically and modulates their conformation. For instance, the refolding of citrate synthase is significantly enhanced by the co-presence of HSP90 (14). The spontaneous refolding of denatured dihydrofolate reductase and irreversible denaturation of firefly luciferase are prevented b...

show abstract

Section: Discussionsupporting

confidence: 64%

Domain Structures and Immunogenic Regions of the 90-kDa Heat-shock Protein (HSP90)

Nemoto

Sato

Iwanari³

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Furthermore, our data suggest that ErbB2/ErbB3 contributes to the branching morphogenesis of SCA cells induced by matrigel. The expression of EGFR (ErbB1) and ErbB2 in HSG cells, which also produce an EGF-like growth factor possessing autocrine regulatory function, has been described [47]. All four EGF receptors are expressed in the submandibular gland.…”

Section: Discussionmentioning

confidence: 98%

Differentiation of a mouse submandibular gland-derived cell line (SCA) grown on matrigel

Barka

Gresik

Miyazaki

2005

Experimental Cell Research

View full text Add to dashboard Cite

“…The supernatant was then applied onto a 10% polyacrylamide gel and electrophoresed. Immunoblotting analysis was performed according to standard procedures (Kyakumoto et al 1994). Specific blots on the membrane were detected with a blotting detection kit utilizing the avidin-biotin alkaline phosphatase complex method (Biomeda, Foster City, Calif.) as described by the supplier.…”

Section: Immunoblotting Analysismentioning

confidence: 99%

“…The adenocarcinoma cell line derived from human salivary gland (HSG) proliferates autonomously, and this proliferation is mediated by an autocrine growth factor, a 46-kDa epidermal growth factor (EGF)-like molecule (Kurokawa et al 1989), and the EGF receptor (Kyakumoto et al 1990(Kyakumoto et al , 1994. The growth of HSG cells is also regulated by several hormones such as glucocorticoids, androgens, and retinoids added exogenously (Kurokawa et al 1988(Kurokawa et al , 1991.…”

Section: Introductionmentioning

confidence: 99%

Expression of retinoid X receptors and COUP-TFI in a human salivary gland adenocarcinoma cell line

Kyakumoto¹,

Nemoto²,

Sato³

et al. 1997

Biochem. Cell Biol.

View full text Add to dashboard Cite

The growth of the adenocarcinoma cell line derived from human salivary gland (HSG) is regulated by all-trans-retinoic acid (t-RA), which binds to its specific receptor, retinoic acid receptors (RARs), located in the nucleus, and thereby transactivates target genes. In this study, we examined the binding characteristics of the nuclear extract of HSG cells to the retinoic acid response element (RARE) compared with those of in vitro translated RAR alpha and retinoid X receptor alpha (RXR alpha), a heterodimeric partner of RAR alpha. Gel shift analysis using anti-RAR alpha and anti-RXR alpha antibodies revealed that the translated RAR alpha bound to RARE as a heterodimer with RXR alpha. In contrast, the binding of the nuclear extract of HSG cells to RARE showed a heterogeneous pattern, suggesting the existence of several species of RXRs as well as RARs in the nuclei of HSG cells. We therefore tried to clone these putative RXRs by the polymerase chain reaction using degenerated oligonucleotide primers conserved across the RXR family. The DNA sequencing of the recombinant clones revealed the expression of RXR alpha and RXR beta. In addition, chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI), which is also an RXR family member, was cloned. To evaluate the transcriptional activity of RARs and RXRs endogenously expressed in HSG cells, we performed a transient transfection analysis. When HSG cells were transfected with a luciferase reporter plasmid containing two repeats of either the RARE of the RAR beta gene or that of cellular retinol-binding protein II gene, positioned upstream of a thymidine kinase promoter fused to the luciferase sequence, a 2-3-fold induction of luciferase activity was observed in both cases. These results suggest that RARs and RXRs endogenously expressed in HSG cells were transcriptionally active in vivo. Thus, our findings showed that RXR alpha, RXR beta, and COUP-TFI are expressed in HSG cells and suggest that these molecules function as heterodimeric partners of RARs and (or) competitive repressors for RAREs and are involved in cellular responses mediated by retinoids.

show abstract

Co-expression of epidermal growth factor-receptor and c-erb B-2 proto-oncogene product in human salivary-gland adenocarcinoma cell line HSG and the implications for HSG cell autocrine growth

Cited by 7 publications

References 35 publications

Domain Structures and Immunogenic Regions of the 90-kDa Heat-shock Protein (HSP90)

Domain Structures and Immunogenic Regions of the 90-kDa Heat-shock Protein (HSP90)

Differentiation of a mouse submandibular gland-derived cell line (SCA) grown on matrigel

Expression of retinoid X receptors and COUP-TFI in a human salivary gland adenocarcinoma cell line

Contact Info

Product

Resources

About