WO1990012869A1 - Cellule eucaryote non melanocytique exprimant de maniere constitutive la tyrosinase humaine biologiquement active, et son utilisation - Google Patents

Cellule eucaryote non melanocytique exprimant de maniere constitutive la tyrosinase humaine biologiquement active, et son utilisation Download PDF

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WO1990012869A1
WO1990012869A1 PCT/US1990/002288 US9002288W WO9012869A1 WO 1990012869 A1 WO1990012869 A1 WO 1990012869A1 US 9002288 W US9002288 W US 9002288W WO 9012869 A1 WO9012869 A1 WO 9012869A1
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tyrosinase
cell
cells
melanocytic
eucaryotic cell
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Brigitte Bouchard
Alan N. Hougton
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Sloan-Kettering Institute For Cancer Research
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/18—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with another compound as one donor, and incorporation of one atom of oxygen (1.14.18)
    • C12Y114/18001—Tyrosinase (1.14.18.1)
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    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Melanocytes and cells of melanocyte lineage are distinguished by their capacity to synthesize the pigment melanin.
  • Melanin production is primarily regulated by the enzyme tyrosinase (monophenol, 3, 4-dihydroxyphenylalanine: oxygen oxidoreductase, EC 1.14.18.1) .
  • melanin synthesis occurs principally in melanosomes which are specialized organelles. Thus, melanin synthesis is generally restricted to melanosome-containing melanocytic cells.
  • This application describes the isolation of a full length of cDNA clone encoding human tyrosinase. Isolation is achieved by using a probe whose sequence is homologous to the Pmel 34 cDNA sequence described by Kwon et al. (1, 35, 37). Moreover, transfection and expression of this new human tyrosinase cDNA clone in mouse fibroblasts induced pigmentation in a cell type which normally does not synthesize melanin. Levels of tyrosinase activity in transfected fibroblasts were equivalent to tyrosinase levels in highly pigmented human melanoma cell lines.
  • tyrosinase-positive fibroblast cell lines demonstrate that melanin synthesis can take place in cells that are not melansome-containing. As a result, we have a tool in which to study the regulation, transport and processing of tyrosinase synthesis.
  • This invention provides a non-melanocytic eucaryotic cell constitutively expressing biologically active human tyrosinase. This invention also provides such a cell which expresses biologically active human tyrosinase under conditions such that the cell produces melanin.
  • this invention provides a method of producing biologically active human tyrosinase and melanin.
  • j Figure 1 Nucleotide and predicated amino acid sequence of BBTY-1 cDNA. The nucleotide sequence is numbered in the 5* to 3' direction. Residues of a predicted signal peptide are indicated by negative numbers, and a cleavage site by a vertical arrow. Termination site (TAA) and polyadenylation signal (869 to 875) are underlined. Potential glycosylation sites are designated by dashed lines.
  • FIG. 1 Northern blot analysis of poly (A + )-selected RNA (4 ⁇ g/lane) from two pigmented melanoma cell lines that express tyrosinase and the B cell lymphoma cell line Daudi. The blot was hybridized with the 3 P-labeled insert of pBBTY- 1. Lanes: 1) SK-MEL-23 melanoma; 2) SK-MEL-19 melanoma; and 3) Daudi.
  • FIG. 1 LpcTYR cells in culture. A nest of cells in the middle of the field contains large, pigmented cytoplasmic granules. Magnification x320.
  • FIG. 1 Tremsmission election micrographs of segments of LpcTYR cells.
  • FIG. 6 Expression of tyrosinase activity in cell extracts from: 1) SK-MEL 19 melanoma; 2) Lpc cells (transfected with pUC 18 plasmid); 3) LpcTYR-1 cells (transfected with BBTY-1 sense construct) 4) LpcTYR-2 cells (transfected with BBTY-1 sense construct) ; and 5) LpcTYW cells (transfected with a BBTY 1 antisense construct) .
  • Tyrosine hydroxylase activity is expressed as (cpm 3 H 2 0/min/mg protein) rl "[bars! or (cpm 3 H 2 0/min/5xl0 6 cells) rI ⁇ V M bars] .
  • FIG. 7 Immunoprecipitation of lysates from 35 S-methionine metabolically labeled SK-MEL-19 melanoma cells, LpcTYR-2 cells expressing BBTY-1, and L929 cells. Lanes: 1) mAb TA99; 2) mAb 2G10; 3) control rabbit sera; and 4) rabbit anti-tyrosinase antisera. A 75 kD band is detected in SK- MEL-19 (with TA99, 2G10 and anti-tyrosinase) and LpcTYR-2 cells (with anti-tyrosinase) . Molecular weight standards: Myosin M chain (200 kD) ; phosphorylase (96 D) ; bovine serum albumin (68 kD) ; and ovalbumin (43 kD) .
  • Figure 8 Indirect immunofluorescence assays for antigen expression by: A) LpcTYR-2 cells expressing BBTY-1, and B) SK-MEL-19 melanoma cells.
  • mAb TA99 anti-gp75
  • mAb CF21 anti-melanoso al antigen •
  • mAb H100-5R28 anti-H-2 k
  • the present invention provides a non-melanocytic eucaryotic cell constitutively expressing biologically active human tyrosinase.
  • biologically active human tyrosinase means a polypeptide having (1) an amino acid sequence identical to, or substantially identical to, the amino acid sequence of and (2) the biological activity of naturally occurring human tyrosinase.
  • non-melanocytic eucaryotic cell means eucaryotic cell characterized by the absence of elanos ⁇ mes, that is, the organelles associated with the production of the polymeric pigment melanin.
  • a fibroblast On example of such a cell is a fibroblast.
  • any eucaryotic cell is useful in the practice - of the subject invention including without limitation, mammalian cells such as human cells for example fibroblast cells, the cells or other animals such as ovine, porcine, murine, bovine or avian cells, insect cells, or yeast cells.
  • Useful non-melanocytic eucaryotic cells include eucaryotic cells in which DNA encoding biologically active human tyrosinase is not naturally present and into which such DNA has been introduced. Methods for introducing such DNA are well known to those skilled in the art as are methods for doing so under conditions such that the DNA will be expressed and biologically active human tyrosinase produced (43).
  • the DNA encoding human tyrosinase is carried on an expression vector suitable for expression in the eucaryotic cell type involved.
  • the expression vector may be a retrovirus; for an insect cell, the expression vector may be a baculovirus.
  • nucleic acid encoding biologically active human tyrosinase may be used in this invention.
  • One such DNA is DNA having the sequence shown in Figure 1 extending from nucleotide 58 to nucleotide 1593 (BBTY-1) .
  • the non-melanocytic eucaryotic cell may be a mammalian cell such as a mouse or human fibroblast cell and may comprise a retroviral expression vector, for example, an expression vector which comprises an SV40 early region promoter and enhancer sequences, an initiation codon immediately upstream of the DNA encoding human tyrosinase, and a termination signal immediately downstream of the DNA encoding human tyrosinase.
  • a retroviral expression vector for example, an expression vector which comprises an SV40 early region promoter and enhancer sequences, an initiation codon immediately upstream of the DNA encoding human tyrosinase, and a termination signal immediately downstream of the DNA encoding human tyrosinase.
  • the non-melanocytic eucaryotic cell may be an insect cell and may comprise a baculovirus expression vector, for example, an expression vector which comprises DNA encoding biologically active human tyrosinase under the expressional control of a polyhydrin promoter (44) .
  • a baculovirus expression vector for example, an expression vector which comprises DNA encoding biologically active human tyrosinase under the expressional control of a polyhydrin promoter (44) .
  • the non-melanocytic eucaryotic cell may be a yeast cell and may comprise an expression plasmid, for example, an expression plasmid which comprises DNA encoding biologically active human tyrosinase under the expressional control of a suitable promoter such as the aleoiiol dehydrogenase isoenzyme I (ADH I) , the phosphoglycerol kinase (PGK) promoter, the repressible acid phosphatase (PH05) promoter and the a factor promoter (plus the signal sequence) .
  • ADH I aleoiiol dehydrogenase isoenzyme I
  • PGK phosphoglycerol kinase
  • PH05 repressible acid phosphatase
  • non- melanocytic eucaryotic cells which constitutively produces biologically active human tyrosinase produces melanin.
  • This invention also provides a method for producing a biologically active human tyrosinase which comprises culturing the cells described hereinabove under conditions such that, the cells express the biologically active human tyrosinase and recovering the human tyrosinase so expressed.
  • Conditions for culturing the cells and for recovering the biologically active human tyrosinase are known in the art and vary depending upon the nature of the eucaryotic cell, expression vector (i any) and the like.
  • This invention also provides a method for producing a biologically active human tyrosinase which comprises culturing the cells described hereinabove under conditions such that the cells express the biologically active human tyrosinase and recovering the human tyrosinase so expressed.
  • a biologically active human tyrosinase which comprises culturing the cells described hereinabove under conditions such that the cells express the biologically active human tyrosinase and recovering the human tyrosinase so expressed.
  • melanin which comprises culturing eucaryotic cells under conditions such that the cells express biologically active human tyrosinase and the tyrosinase so expressed catalyzes the production of melanin, and then recovering the melanin so produced.
  • TK " L929 cells (mouse fibroblast) (3) were used for transfection experiments.
  • Cell lines were maintained in Eagle's Minimum Essential Medium supplemented with 2 mM glutamine, O.lmM non-essential amino acids, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 10% fetal bovine serum (complete medium) .
  • Cells were passaged with trypsin (1 mg/ml and EDTA (0.2 mg/ml). All cultures were checked regularly for the presence of mycoplasma and contaminated cultures were discarded.
  • Cell pellets were fixed in Karnofsky's fixative overnight, rinsed in PBS for 1 hour, then post-fixed for 1 hour in 1% osmium tetroxide-PBS solution.
  • Cell pellets were dehydrated in graded ethyl alcohol followed by propylene oxide, and embedded in Maraglas-D.E.R. 732 epoxy resin (Dow Chemical Co., Midland, Michigan).
  • 1 ⁇ m thick sections were stained with borate-buffered 1% toluidine blue. Thin sections were stained with uranyl acetate followed by lead citrate and examined with a Philips 410 LS electron microscope.
  • cpNJ Library cfflfl $creepjpg t A cDNA library was constructed from 3 ⁇ g of poly(A+) selected mRNA (4) prepared from the human melanotic melanoma cell line S_. ⁇ -MEL-19 (2) . Full length cDNA was synthesized, rendered blunt-ended using Klenow enzyme, and tailed with EcoRI linkers (New England Biolabs, Inc., Beverly, MA) (5). The cDNA was then size-fractionated on Ultrogel Aca 34 (Pharmacia Fine Chemicals, Piscataway, NJ) (6).
  • cDNA molecules > 800 bp were used to construct a library of 3 x 10 5 recombinants in the lambda phage vector gtl0(7).
  • a 50 base oligonucleotide probe 50-mer, shown below
  • a 50 base oligonucleotide probe 50-mer, shown below
  • the oligonucleotide was synthesized on an Applied Biosystem DNA synthesizer, model 310 A (Applied Biosystems, Foster City, CA) .
  • the probe was end-labeled with gamma 3 P-ATP and T4 polynucleotide kinase (4) .
  • Prehybridization and hybridization were carried out at 48°C for 4 hours and 18 hours, respectively, in 6x NET (lx NET is 0.15M NaCl, l mM EDTA, and 15mM Tris-HCl, pH 8) , 0.1% SDS and 5x Denhardt's solution (0.1% BSA, 0.1% Ficoll 400, 0.1% polyvinyl- pyrolidone) , and 100 ⁇ g/ml of denatured salmon sperm DNA.
  • Duplicate filters were washed in 6x NET, 0.1% SDS at room temperature, followed by stringent washes at 55° and 60°C. The filters were then autoradiographed for 4 hours at -70°.
  • Plaque purified phage DNA was restricted with EcoRI and cDNA inserts were subcloned into the plasmid vector pUC 18(8). Recombinant plasmids and deletion subclones subsequently obtained by digestion with exonuclease IIl/Mung Bean nuclease (9) were sequenced by the dideoxynucleotide chain termination method (10) . Northern Blot Analysis.
  • Poly (A+)-mRNA (4 ⁇ g) was fractioned on 1% formaldehyde denaturing agarose gels (4) , transferred of Gene Screen Plus membranes (Dupont, New England Nuclear, Boston, MA) , and hybridized to a ⁇ P-labeled cDNA probe.
  • the filters were washed twice at room temperature in 2x SSC (IX SSC is 0.15M NaCl, 0.015 M sodium citrate pH 7) and 1% SDS, then stringent washes were carried out at 55 ⁇ C in IX SSC, 1% SDS, and at 65°C in 0.1X SSC, 1% SDS, for 15 minutes each.
  • the cDNA inserts were subcloned into the EcoRI site of the expression vector pcEXV-3, which allows expression of cDNA under the control of SV40 early region promotor and enhancer sequences (11) .
  • Expression plasmids containing cDNA inserts in opposite orientations (5'—> 3' or 3'—> 5') were constructed.
  • Sense and antisense oriented plasmids were designated pcTYR and pcTYW, respectively.
  • L929 cells were cotransfected by the calcium phosphate precipitation technique (12) with the following combinations: (1) transfection with pUC 18, pSV2 neo plasmid, and high molecular weight carrier DNA from L929 cells; (2) transfection with pcTYR, pSV2 neo plasmid, and high molecular weight carrier DNA from L929 cells; or (3) transfection with pcTYR, pSV2 neo plasmid, and high molecular weight carrier DNA from L929 cells.
  • transfectants were started on day 3 following transfection with 1 mg/ml of the antibiotic G418 (Sigma Chemical Co., St. Louis, MO) . Complete medium with G418 was replaced every 3 days and colonies appearing on days 10-14 were isolated using cloning rings and expanded.
  • the mouse origin of transfected cell lines was confirmed by positive anti-mouse Ig mixed hemadsorption assays using H-100-5R28, a monoclonal antibody directed against H-2K k (mouse MHC class I antigens) (13), and lack of reactivity with mAb M3- 68 (14) or AJ2 (15) which recognize virtually all human melanoma cells but not L929 cells.
  • CF21 (igGl) and TA 9 (IgG2a) are mAb which have been previously described that recognize distinct antigens in human melanosomes (16) .
  • the monoclonal antibody 2G10 (IgG2a) recognizes a 75 kD intracellular glycoprotein of pigmented melanotic cells (17) .
  • mAb AJ2 (IgGl) recognizes the beta subunit of human VLA/integrin molecules (15, 18).
  • Rabbit anti-tyrosinase antiserum was raised by immunization with purified mouse tyrosinase (19) .
  • Tyrosinase was purified by DEAE ion exchange chromatography followed by sequential discontinuouspolyacrylamidegelelectrophoresis.
  • Anti-mouse immunoglobulin hemadsorption assays and indirect immunofluorescence studies were performed as described (2, 20) .
  • cDNA clones were isolated from a lambda gtlO library derived from the pigmented human melanoma cell line SK-MEL-19 (see Materials and Methods) . 10 s recombinant cDNA clones were screened and four reactive clones were plaque purified. The four cDNA inserts were subcloned into the plasmid vector pUC 18 and clones were designated pBBTY-1, -2, -3, and -4.
  • pBBTY-1 and pBBTY-2 Two clones, pBBTY-1 and pBBTY-2, each containing cDNA inserts of 2 kb, had restriction maps identical to each other and to that of Pmel 34 reported by Kwon et al. (1) (digested with Bgl II, Hpa II, Msp I, Neo I, Pvu II, and Taq I).
  • the cDNA inserts in clones pBBTY-3 and pBBTY-4 were 1.7 and 1.8 kb, respectively.
  • the restriction map of pBBTY-3 was different from those of pBBTY-1 and pBBTY-2 downstream of position 960 (a PvuII restriction site) .
  • pBBTY-1 was subsequently sequenced and used for further experiments.
  • the nucleotide sequence of BBTY-1 (Fig. 1) contained a single open reading frame of 1593 residues capable of encoding a 531 amino acid (aa) polypeptide with a derived molecular weight of 60.37 kD. A leader peptide of 19 aa was assigned to positions -19 through -1 (23). The processed core protein was predicted to have a molecular weight of 58.118 kD. Seven potential N-glycosylation signals (Asn-X- Ser/Thr) were predicted at positions 69, 94, 144, 213, 273, 320, and 354.
  • BBTY-1 was a candidate for a full length cDNA clone encompassing a complete coding region.
  • BBTY-1 cDNA was used to detect mRNA transcripts in Northern blot analysis of a panel of melanoma cell lines, including those known to express tyrosinase activity as well as tyrosinase-negative melanomas.
  • the major transcript detected was 2.4 kb, but a weaker signal was seen at 4.7 kb ( Figure 2) .
  • BBTY-l was transfected into L929 mouse fibroblasts using the expression vector pcEXV-3 (11) .
  • L929 cells transfected with pcTYR sense orientation
  • Control cells transfected with pcTYW antisense orientation
  • LpcTYW antisense orientation
  • LpC plasmid pUC 18
  • LpcTYR cells contained pigment, while no pigmentation was detected in LpcTYW, LpC or untransfected L929 cells.
  • the cell pellets of LpcTYR clones were dark brown in contrast to the non-pigmented pellets of LpcTYW and LpC cultures.
  • LpcTYR Cell pellets of LpcTYR were more deeply pigmented when cultures were harvested at confluency. LpcTYR clones have continued to produce pigment for more than 5 months in continuous culture.
  • absorption spectra of cell extracts from LpcTYR and control L929 cells were compared to those of extracts of the pigmented melanoma cell line SK-MEL-19 and purified melanin.
  • LpcTYR and SK- MEL-19 extracts and melanin had identical patterns of absorption, with broad absorption from 360 nm to >450 nm; this absorption pattern was not observed with L929 cell extracts.
  • the absorption patterns by extracts of LpcTYR and SK-MEL-19 and melanin standard were identical to the previously described absorption spectra for melanin (26) .
  • LpcTYR cells but not control LpC cells, had cytoplasmic membrane-bound vesicles (Fig. 5) containing electron dense material consistent with melanin. There was no evidence of melanosomal structural elements within LpcTYR cells or Lpc cells.
  • tyrosine hydroxylase activity was measured in protein extracts of subclones of LpcTYR, LpcTYW and LpC.
  • extracts of LpcTYW and LpC contained no detectable tyrosinase activity.
  • ⁇ _ LpcTYR-2, SK-MEL-19 melanoma cells and control L929 cells were metabolically labeled with 35 S-methionine and cell extractswere immunoprecipitatedwithrabbit anti-tyrosinase antiserum or mAb TA99 (which detects the melanosomal antigen gp75) .
  • mAb 2G10 which is also directed against an intracellular 75 kD antigen expressed by pigmented melanoma cells (17) was tested.
  • Anti-tyrosinase antiserum detected a 75 kD protein in LpcTYR-2 cells and a protein of the same size in SK-MEL-19 melanoma cells (Fig. 7) .
  • the molecular size of tyrosinase in LpcTYR-2 and SK-MEL-19 cells corresponded to the size of glycosylated tyrosinase.
  • a very faint band at approximately 75 kD was inconsistently detected in L929 cells with anti-tyrosinase antiserum - this likely represents a cross-reaction of poly ⁇ lonal sera to a non-tyrosinase molecule in L929 cells since no tyrosinase activity or tyrosinase transcript was detected in these cells and cold lysates from L929 cells did not block immunoprecipitation of tyrosinase from LpcTYR-2.
  • Tyrosinase catalyzes the o-hydroylyation of monophenols and oxidation of o.-diphenols to o_-guinones.
  • tyrosinase enzymatically converts tyrosine to dihydroxyphenylalanine (DOPA) and DOPA to dopaquinone, leading to the spontaneous formation of the complex mixture of pigments known as melanin (27) .
  • DOPA dihydroxyphenylalanine
  • melanin melanin
  • the later steps in this pathway are not well characterized, and it has been suggested that a number of other factors, both catalytic and inhibitory, may regulate melanin synthesis and influence the species of melanin formed (28, 29).
  • the complexity of pigment expression has been further highlighted by genetic studies in the mouse where more than 50 loci have been found to influence coat color (30) . Thus, it is possible that a number of gene products, most not yet identified, can play a role in elanogenesis.
  • transfected L929 fibroblasts not only stably expressed tyrosinase activity but were able to produce and package melanin.
  • Melanin precursors are cytotoxic, and it has been presumed that melanocytic cells containmechanisms, perhaps located within melanosomes, that protect from the effects of toxic intermediates.
  • Melanin precursors were cytotoxic in transfected L929 cells, and that cells producing substantial amounts of pigment were destined to die, based on the following observations: (1) only a subpopulation of transfected cells contained pigmented vesicles; (2) deeply pigmented, non-viable cells were observed floating in the supernatant of transfectant cultures; and (3) when transfected cells were cryopreserved and then thawed, pigmented cells were not initially detected but eventually repopulated the culture.
  • human tyrosinase is glycosylated to a form identical in size to fully processed tyrosinase expressed in human melanocytic cells. It is likely that human tyrosinase was processed through the Golgi apparatus in L929 cells and transported to or remaining in vesicles arising from the trans Golgi. The nature and destination of these vesicles is not known. It is interesting to speculate that these vesicles might be precursors of melanosomes but that formation of melanosomes would depend on the products of other specialized genes.
  • tyrosinase The expression and regulation of tyrosinase has been the subject of extensive studies, but the formal identification of the gene that codes for tyrosinase has not been straightforward (reviewed in 31) .
  • Two distinct, and distantly related, genes have been proposed as candidates for mouse tyrosinase, based on detection of mRNA of these genes in melanocytic cells and reactivity of the protein product with antibodies against tyrosinase (32, 33). Neither gene, however, was demonstrated to directly code for a product with tyrosinase activity.
  • the Pmel 34 cDNA clone was detected by screening a cDNA library with polyclonal antisera raised against hamster tyrosinase.
  • Pmel 34 has - ⁇ . been mapped to the c-albino locus in the mouse, the presumed site of the tyrosinase structural gene or a gene that regulates tyrosinase expression.
  • the nucleotide and predicted aa sequences of BBTY-1 and Pmel 34 are closely similar, except BBTY-1 contains an initiation codon that is not present in Pmel 34. Additionally, there are minor differences in nucleotide and predicted aa sequences.
  • transcripts of the tyrosinase gene have been found . in mouse melanoma cells (36) .
  • the remaining transcripts are generated by alternative splicing leading to deletion of internal sequences, presumably by exon skipping or by selection of internal splice sites.
  • these alternative transcripts have been expressed, they have not been found to encode active tyrosinase (34, 36).
  • the BBTY-1 cDNA represents the human counterpart of the mouse pmcTyrl transcript.
  • Another cDNA clone which was isolated, BBTY-3 differs from BBTY-1 in its 3* restriction map, possibly corresponding to an alternative transcript of the human tyrosinase gene.
  • mAb 2G10 immunodepletes tyrosinase activity (37) and therefore possibly recognizes a molecule with tyrosinase activity.
  • mAb 2G10 did not react with human tyrosinase encoded by BBTY-1, suggesting that mAb 2G10 recognizes a distinct molecule from the gene product of BBTY-1.
  • TA99 mAb recognizes an acidic 75 kD glycoprotein (38) , and the antigen recognized by TA99 is a candidate for tyrosinase, based on its expression in melanosomes, its molecular size and charge.
  • the finding that mAb TA99 and CF21 did not react with L929 transfectants provides evidence that they do not recognize determinants coded for by the BBTY-1 human tyrosinase molecule.
  • mAb TA99 does not recognize tyrosinase: 1) mAb TA99 does not precipitate tyrosinase activity from melanoma cell extracts (39, 40); 2) the TA99 antigen, gp75, is generally co- expressed with tyrosinase activity, but there are examples of gp75* melanoma cell lines that do note express tyrosinase activity; and 3) the expression of tyrosinase and gp75 in melanoma cell lines has been independently regulated (20) .
  • a distinguishing characteristic of cells of the melanocyte lineage is the expression of the melanosomal enzyme tyrosinase which- catalyzes the synthesis of the pigment melanin.
  • a tyrosinase cDNA clone, designated BBTY-1 was isolated from a library constructed from the pigmented TA99 + /CF21 + melanoma cell line SK-MEL-19, Expression of BBTY-1 in mouse L929 fibroblasts led to synthesis and expression of active tyrosinase, and, unexpectedly, to stable production of melanin. Melanin was synthesized and stored within membrane-bound vesicles in the cytoplasm of transfected fibroblasts.
  • BBTY-1 detected a 2.4 kb mRNA transcript in 9 of 9 pigmented, tyrosinase-positive melanoma cell lines. Tyrosinase transcripts of the same size were detected in a subset (3 of 8) of non-pigmented, tyrosinase- negative .melanoma cell lines, suggesting that post- transcriptional events are important in regulating tyrosinase activity.

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Abstract

Cette invention concerne une cellule eucaryote non melanocytique exprimant de manière constitutive la tyrosinase humaine biologiquement active. La présente invention concerne également des procédés de production de tyrosinase humaine biologiquement active. De plus, l'invention concerne une cellule eucaryote non mélanocytique qui exprime de manière constitutive la tyrosinase humaine biologiquement active qui, à son tour, catalyse la production de mélanine. La mélanine ainsi produite peut alors être récupérée.
PCT/US1990/002288 1989-04-26 1990-04-26 Cellule eucaryote non melanocytique exprimant de maniere constitutive la tyrosinase humaine biologiquement active, et son utilisation WO1990012869A1 (fr)

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US34396089A 1989-04-26 1989-04-26
US343,960 1989-04-26

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EP (1) EP0507772A1 (fr)
JP (1) JPH04505249A (fr)
CA (1) CA2054709A1 (fr)
WO (1) WO1990012869A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0687180A1 (fr) * 1992-12-22 1995-12-20 Ludwig Institute For Cancer Research Procedes de detection et de traitement de personnes presentant des cellules anormales exprimant les antigenes peptidiques hla-a2/tyrosinase
EP0711173A1 (fr) * 1993-06-17 1996-05-15 Ludwig Institute For Cancer Research Peptides isoles formant des complexes avec la molecule mhc du clone hla-c 10 et utilisation de ceux-ci
EP1025849A1 (fr) * 1992-12-22 2000-08-09 Ludwig Institute For Cancer Research Procedes de detection et de traitement de personnes presentant des cellules anormales exprimant les antigenes peptidiques hla-a2/tyrosinase
EP1096021A2 (fr) * 1997-02-06 2001-05-02 Biosource Technologies, Inc. Melanines a capacité ameliorée d'inhibition de la replication du HIV
WO2001070792A1 (fr) * 2000-03-07 2001-09-27 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, tyrosinase humaine 12, et polynucleotide codant pour ce polypeptide
WO2014016118A1 (fr) 2012-07-26 2014-01-30 Unilever N.V. Méthode de préparation d'une tyrosinase humaine recombinée

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR9910313B1 (pt) 1998-05-08 2008-11-18 reservatàrio de sangue/permutador tÉrmico combinado.
TW200716979A (en) * 1999-06-29 2007-05-01 Univ New York Screening methods for compounds that affect melanogenesis

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4898814A (en) * 1986-10-06 1990-02-06 Donald Guthrie Foundation For Medical Research, Inc. A cDNA clone for human tyrosinase

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Biochemical and Biophysical Research Communications, Volume 162 No. (3), issued 15 August 1989, TAKEDA et al, "Functional Analysis of the cDNA encoding human tyrosinase precursor" pages 984-990. See entire article. *
EMBO, Volume 7 (9), issued September 1988, MULLER et al, "Functional analysis of alternatively spliced tyrosinase gene transcripts", pages 2723-2730. See entire article. *
Journal of Experimental Medicine, Volume 169, issued June 1989, BOUCHARD et al, "Induction of pigmentation in Mouse Fibroblasts by expression of human tyrosinase cDNA", pages 2029-2042. See entire article. *
Nucleic Acids Research, Volume 14 (6), issued 26 February 1986, SHIBAHARA et al, "Cloning and expression of cDNA encoding mouse tyrosinase", pages 2413-2427. See entire article. *
See also references of EP0507772A4 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0687180A1 (fr) * 1992-12-22 1995-12-20 Ludwig Institute For Cancer Research Procedes de detection et de traitement de personnes presentant des cellules anormales exprimant les antigenes peptidiques hla-a2/tyrosinase
EP0687180A4 (fr) * 1992-12-22 1998-01-07 Ludwig Inst Cancer Res Procedes de detection et de traitement de personnes presentant des cellules anormales exprimant les antigenes peptidiques hla-a2/tyrosinase
EP1025849A1 (fr) * 1992-12-22 2000-08-09 Ludwig Institute For Cancer Research Procedes de detection et de traitement de personnes presentant des cellules anormales exprimant les antigenes peptidiques hla-a2/tyrosinase
EP0711173A1 (fr) * 1993-06-17 1996-05-15 Ludwig Institute For Cancer Research Peptides isoles formant des complexes avec la molecule mhc du clone hla-c 10 et utilisation de ceux-ci
EP0711173A4 (fr) * 1993-06-17 1998-04-15 Ludwig Inst Cancer Res Peptides isoles formant des complexes avec la molecule mhc du clone hla-c 10 et utilisation de ceux-ci
EP1096021A2 (fr) * 1997-02-06 2001-05-02 Biosource Technologies, Inc. Melanines a capacité ameliorée d'inhibition de la replication du HIV
EP1096021A3 (fr) * 1997-02-06 2004-03-10 Large Scale Biology Corporation Mélanines à capacité améliorée d'inhibition de la réplication du HIV
WO2001070792A1 (fr) * 2000-03-07 2001-09-27 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, tyrosinase humaine 12, et polynucleotide codant pour ce polypeptide
WO2014016118A1 (fr) 2012-07-26 2014-01-30 Unilever N.V. Méthode de préparation d'une tyrosinase humaine recombinée
EA028538B1 (ru) * 2012-07-26 2017-11-30 Юнилевер Н.В. Способ изготовления рекомбинантной тирозиназы человека

Also Published As

Publication number Publication date
EP0507772A1 (fr) 1992-10-14
EP0507772A4 (fr) 1992-06-11
JPH04505249A (ja) 1992-09-17
CA2054709A1 (fr) 1990-10-27

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