b6637a239ce95d3ab1bd3c89fac2eef8 cancers-13-01869.pdf bc2334b54ceed4e717835beef1527a8858e942b4 cancers-13-01869.pdf e5883914025da5392a399c7e91029bcfa1140a32f7f73f6c7637bf53eb275235 cancers-13-01869.pdf Title: Gene Amplification-Associated Overexpression of the Selenoprotein tRNA Enzyme TRIT1 Confers Sensitivity to Arsenic Trioxide in Small-Cell Lung Cancer Subject: The alteration of RNA modification patterns is emerging as a common feature of human malignancies. If these changes affect key RNA molecules for mRNA translation, such as transfer RNA, they can have important consequences for cell transformation. TRIT1 is the enzyme responsible for the hypermodification of adenosine 37 in the anticodon region of human tRNAs containing serine and selenocysteine. Herein, we show that TRIT1 undergoes gene amplification-associated overexpression in cancer cell lines and primary samples of small-cell lung cancer. From growth and functional standpoints, the induced depletion of TRIT1 expression in amplified cells reduces their tumorigenic potential and downregulates the selenoprotein transcripts. We observed that TRIT1-amplified cells are sensitive to arsenic trioxide, a compound that regulates selenoproteins, whereas reduction of TRIT1 levels confers loss of sensitivity to the drug. Overall, our results indicate a role for TRIT1 as a small-cell lung cancer-relevant gene that, when undergoing gene amplification-associated activation, can be targeted with the differentiation agent arsenic trioxide. Keywords: small-cell lung cancer; transfer RNA; RNA modifications; TRIT1; gene amplification; selenoproteins Author: Laia Coll-SanMartin, Veronica Davalos, David Piñeyro, Margalida Rosselló-Tortella, Alberto Bueno-Costa, Fernando Setien, Alberto Villanueva, Isabel Granada, Neus Ruiz-Xiviller, Annika Kotter, Mark Helm, Jun Yokota, Reika Kawabata-Iwakawa, Takashi Kohno and Manel Esteller Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Apr 14 10:02:36 2021 CEST ModDate: Wed Apr 14 10:07:54 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1436946 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OGLZWV+VnURWPalladioL Type 1 Custom yes yes no 10 0 OFHLMU+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 LAGIGW+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 JDFPFY+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 SOZKOC+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 LCJEVJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 37 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 59 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 63 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 67 0 TELXLI+CMSY10 Type 1 Builtin yes yes no 79 0 PTVOAX+EURM10 Type 1 Builtin yes yes no 86 0 CCNLFE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 102 0 CCNLFG+PalatinoLinotype TrueType WinAnsi yes yes no 105 0 CCNLFI+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 108 0 CCNLFE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 122 0 CCNLFG+PalatinoLinotype TrueType WinAnsi yes yes no 125 0 CCNLFI+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 128 0 QLBIEB+URWPalladioL-BoldItal Type 1 Custom yes yes no 136 0 CCNLFE+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 146 0 CCNLFG+PalatinoLinotype TrueType WinAnsi yes yes no 149 0 CCNLFI+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 152 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-06-30 02:39:02 CEST RepresentationInformation: cancers-13-01869.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-01-24 19:57:35 CET Size: 1436946 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 375 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Human Cell Lines Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Human Biological Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Fluorescence In Situ Hybridization (FISH) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Multiplex Ligation-Dependent Probe Amplification (MLPA) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: TRIT1 Short-Hairpin RNA Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Western Blot Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: N6-Isopentenyladenosine (I6A) Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Murine Models Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Drug-Dose Response Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: RNA Sequencing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Children: Item: Title: Detection of TRIT1 Gene Amplification-Associated Overexpression in Small-Cell Lung Cancer Cell Lines Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Cellular and Molecular Effects of TRIT1 Depletion in Gene Amplified Small-Cell Lung Cancer Cells Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Occurrence of TRIT1 Gene Amplification in Small-Cell Lung Cancer Patients and In Vitro and In Vivo Sensitivity to Arsenic Trioxide Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Info: Title: Gene Amplification-Associated Overexpression of the Selenoprotein tRNA Enzyme TRIT1 Confers Sensitivity to Arsenic Trioxide in Small-Cell Lung Cancer Author: Laia Coll-SanMartin, Veronica Davalos, David Piñeyro, Margalida Rosselló-Tortella, Alberto Bueno-Costa, Fernando Setien, Alberto Villanueva, Isabel Granada, Neus Ruiz-Xiviller, Annika Kotter, Mark Helm, Jun Yokota, Reika Kawabata-Iwakawa, Takashi Kohno and Manel Esteller Subject: The alteration of RNA modification patterns is emerging as a common feature of human malignancies. If these changes affect key RNA molecules for mRNA translation, such as transfer RNA, they can have important consequences for cell transformation. TRIT1 is the enzyme responsible for the hypermodification of adenosine 37 in the anticodon region of human tRNAs containing serine and selenocysteine. Herein, we show that TRIT1 undergoes gene amplification-associated overexpression in cancer cell lines and primary samples of small-cell lung cancer. From growth and functional standpoints, the induced depletion of TRIT1 expression in amplified cells reduces their tumorigenic potential and downregulates the selenoprotein transcripts. We observed that TRIT1-amplified cells are sensitive to arsenic trioxide, a compound that regulates selenoproteins, whereas reduction of TRIT1 levels confers loss of sensitivity to the drug. Overall, our results indicate a role for TRIT1 as a small-cell lung cancer-relevant gene that, when undergoing gene amplification-associated activation, can be targeted with the differentiation agent arsenic trioxide. Keywords: small-cell lung cancer; transfer RNA; RNA modifications; TRIT1; gene amplification; selenoproteins Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Apr 14 10:02:36 CEST 2021 ModDate: Wed Apr 14 10:07:54 CEST 2021 ID: 0x4a7e5538b476ba8e328e955875bf6701, 0x4a7e5538b476ba8e328e955875bf6701 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: LAGIGW+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: LAGIGW+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: PTVOAX+EURM10 FontSubset: true FirstChar: 12 LastChar: 25 FontDescriptor: FontName: PTVOAX+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: JDFPFY+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: JDFPFY+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: QLBIEB+URWPalladioL-BoldItal FontSubset: true FirstChar: 110 LastChar: 112 FontDescriptor: FontName: QLBIEB+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OGLZWV+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: OGLZWV+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: SOZKOC+VnURWPalladioL-Bold FontSubset: true FirstChar: 226 LastChar: 226 FontDescriptor: FontName: SOZKOC+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OFHLMU+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: OFHLMU+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: TELXLI+CMSY10 FontSubset: true FirstChar: 2 LastChar: 48 FontDescriptor: FontName: TELXLI+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 153, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 135, 641, 146, 654 Annotation: Subtype: Link Rect: 235, 641, 246, 654 Annotation: Subtype: Link Rect: 328, 641, 339, 654 Annotation: Subtype: Link Rect: 136, 629, 147, 642 Annotation: Subtype: Link Rect: 171, 616, 182, 629 Annotation: Subtype: Link Rect: 380, 616, 391, 629 Annotation: Subtype: Link Rect: 65, 346, 154, 355 Annotation: Subtype: Link Rect: 35, 336, 87, 343 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 114, 17, 265, 28 Annotation: Subtype: Link Rect: 408, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 482, 87, 501 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 311, 608, 318, 617 ActionDest: 163 Annotation: Subtype: Link Rect: 321, 608, 328, 617 ActionDest: 163 Annotation: Subtype: Link Rect: 199, 558, 206, 567 ActionDest: 163 Annotation: Subtype: Link Rect: 209, 557, 216, 567 ActionDest: 163 Annotation: Subtype: Link Rect: 480, 533, 487, 541 ActionDest: 163 Annotation: Subtype: Link Rect: 488, 532, 495, 541 ActionDest: 163 Annotation: Subtype: Link Rect: 286, 507, 293, 516 ActionDest: 163 Annotation: Subtype: Link Rect: 296, 507, 303, 516 ActionDest: 163 Annotation: Subtype: Link Rect: 440, 470, 447, 479 ActionDest: 163 Annotation: Subtype: Link Rect: 450, 470, 462, 479 ActionDest: 163 Annotation: Subtype: Link Rect: 303, 444, 315, 454 ActionDest: 163 Annotation: Subtype: Link Rect: 318, 445, 330, 454 ActionDest: 163 Annotation: Subtype: Link Rect: 477, 432, 489, 441 ActionDest: 163 Annotation: Subtype: Link Rect: 491, 432, 503, 441 ActionDest: 163 Annotation: Subtype: Link Rect: 351, 407, 363, 416 ActionDest: 163 Annotation: Subtype: Link Rect: 212, 382, 224, 391 ActionDest: 163 Annotation: Subtype: Link Rect: 439, 357, 451, 366 ActionDest: 163 Annotation: Subtype: Link Rect: 452, 357, 464, 366 ActionDest: 163 Annotation: Subtype: Link Rect: 505, 344, 512, 353 ActionDest: 163 Annotation: Subtype: Link Rect: 513, 344, 525, 353 ActionDest: 163 Page: Sequence: 3 Page: Sequence: 4 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 344, 493, 529, 505 Annotation: Subtype: Link Rect: 361, 406, 369, 418 ActionDest: 96 Annotation: Subtype: Link Rect: 432, 384, 444, 393 ActionDest: 166 Annotation: Subtype: Link Rect: 445, 384, 457, 393 ActionDest: 166 Annotation: Subtype: Link Rect: 349, 331, 357, 343 ActionDest: 96 Annotation: Subtype: Link Rect: 544, 309, 556, 318 ActionDest: 166 Annotation: Subtype: Link Rect: 251, 281, 258, 293 ActionDest: 96 Page: Sequence: 6 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 184, 735, 196, 744 ActionDest: 166 Annotation: Subtype: Link Rect: 197, 735, 209, 744 ActionDest: 166 Annotation: Subtype: Link Rect: 363, 707, 370, 719 ActionDest: 96 Annotation: Subtype: Link Rect: 362, 657, 369, 669 ActionDest: 96 Annotation: Subtype: Link Rect: 524, 632, 531, 644 ActionDest: 96 Annotation: Subtype: Link Rect: 541, 521, 548, 533 ActionDest: 116 Annotation: Subtype: Link Rect: 408, 458, 415, 470 ActionDest: 116 Annotation: Subtype: Link Rect: 371, 423, 383, 432 ActionDest: 166 Annotation: Subtype: Link Rect: 200, 395, 207, 407 ActionDest: 116 Annotation: Subtype: Link Rect: 463, 398, 475, 407 ActionDest: 163 Annotation: Subtype: Link Rect: 478, 398, 490, 407 ActionDest: 163 Annotation: Subtype: Link Rect: 353, 269, 360, 281 ActionDest: 116 Annotation: Subtype: Link Rect: 415, 234, 422, 244 ActionDest: 163 Annotation: Subtype: Link Rect: 422, 235, 434, 244 ActionDest: 163 Annotation: Subtype: Link Rect: 467, 182, 474, 194 ActionDest: 116 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 447, 173, 454, 185 ActionDest: 133 Annotation: Subtype: Link Rect: 421, 161, 428, 173 ActionDest: 140 Annotation: Subtype: Link Rect: 549, 148, 556, 160 ActionDest: 133 Annotation: Subtype: Link Rect: 524, 126, 536, 135 ActionDest: 166 Annotation: Subtype: Link Rect: 396, 76, 408, 85 ActionDest: 166 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 282, 720, 289, 732 ActionDest: 140 Annotation: Subtype: Link Rect: 510, 710, 522, 719 ActionDest: 166 Annotation: Subtype: Link Rect: 201, 672, 213, 681 ActionDest: 166 Annotation: Subtype: Link Rect: 402, 647, 414, 656 ActionDest: 166 Annotation: Subtype: Link Rect: 393, 609, 405, 618 ActionDest: 166 Annotation: Subtype: Link Rect: 404, 262, 416, 271 ActionDest: 166 Annotation: Subtype: Link Rect: 417, 262, 429, 271 ActionDest: 166 Annotation: Subtype: Link Rect: 426, 237, 438, 246 ActionDest: 166 Annotation: Subtype: Link Rect: 438, 237, 450, 246 ActionDest: 166 Annotation: Subtype: Link Rect: 413, 225, 425, 234 ActionDest: 166 Annotation: Subtype: Link Rect: 408, 212, 420, 221 ActionDest: 166 Annotation: Subtype: Link Rect: 420, 212, 432, 221 ActionDest: 166 Annotation: Subtype: Link Rect: 544, 212, 556, 221 ActionDest: 166 Annotation: Subtype: Link Rect: 190, 187, 202, 196 ActionDest: 166 Annotation: Subtype: Link Rect: 309, 172, 316, 184 ActionDest: 116 Annotation: Subtype: Link Rect: 303, 149, 315, 158 ActionDest: 166 Annotation: Subtype: Link Rect: 545, 149, 557, 158 ActionDest: 166 Annotation: Subtype: Link Rect: 231, 137, 243, 146 ActionDest: 166 Annotation: Subtype: Link Rect: 303, 124, 315, 133 ActionDest: 166 Annotation: Subtype: Link Rect: 378, 59, 385, 71 ActionDest: 140 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 242, 720, 249, 732 ActionDest: 140 Annotation: Subtype: Link Rect: 199, 632, 206, 644 ActionDest: 140 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 479, 707, 491, 716 ActionDest: 166 Annotation: Subtype: Link Rect: 492, 707, 504, 716 ActionDest: 166 Annotation: Subtype: Link Rect: 349, 656, 361, 666 ActionDest: 166 Annotation: Subtype: Link Rect: 361, 656, 373, 666 ActionDest: 166 Annotation: Subtype: Link Rect: 370, 606, 382, 615 ActionDest: 166 Annotation: Subtype: Link Rect: 379, 493, 391, 502 ActionDest: 166 Annotation: Subtype: Link Rect: 391, 493, 403, 502 ActionDest: 166 Annotation: Subtype: Link Rect: 515, 468, 527, 477 ActionDest: 166 Annotation: Subtype: Link Rect: 333, 430, 345, 440 ActionDest: 166 Annotation: Subtype: Link Rect: 345, 430, 357, 440 ActionDest: 166 Annotation: Subtype: Link Rect: 391, 406, 403, 414 ActionDest: 166 Annotation: Subtype: Link Rect: 290, 393, 302, 402 ActionDest: 166 Annotation: Subtype: Link Rect: 277, 355, 289, 364 ActionDest: 166 Annotation: Subtype: Link Rect: 245, 292, 257, 301 ActionDest: 166 Annotation: Subtype: Link Rect: 529, 217, 536, 226 ActionDest: 163 Annotation: Subtype: Link Rect: 536, 217, 543, 226 ActionDest: 163 Annotation: Subtype: Link Rect: 363, 154, 375, 163 ActionDest: 166 Annotation: Subtype: Link Rect: 376, 154, 388, 163 ActionDest: 166 Annotation: Subtype: Link Rect: 529, 129, 541, 138 ActionDest: 166 Annotation: Subtype: Link Rect: 323, 104, 334, 113 ActionDest: 166 Annotation: Subtype: Link Rect: 381, 92, 393, 101 ActionDest: 166 Annotation: Subtype: Link Rect: 394, 92, 405, 101 ActionDest: 166 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 221, 722, 233, 731 ActionDest: 166 Annotation: Subtype: Link Rect: 234, 722, 246, 731 ActionDest: 166 Annotation: Subtype: Link Rect: 531, 710, 543, 719 ActionDest: 169 Annotation: Subtype: Link Rect: 544, 710, 556, 719 ActionDest: 169 Annotation: Subtype: Link Rect: 298, 672, 310, 681 ActionDest: 169 Annotation: Subtype: Link Rect: 419, 495, 560, 506 Annotation: Subtype: Link Rect: 166, 483, 274, 494 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 381, 650, 560, 661 Annotation: Subtype: Link Rect: 166, 638, 223, 649 Annotation: Subtype: Link Rect: 495, 543, 532, 554 Annotation: Subtype: Link Rect: 451, 532, 488, 542 Annotation: Subtype: Link Rect: 495, 532, 531, 542 Annotation: Subtype: Link Rect: 147, 509, 185, 519 Annotation: Subtype: Link Rect: 239, 485, 277, 496 Annotation: Subtype: Link Rect: 283, 485, 319, 496 Annotation: Subtype: Link Rect: 450, 474, 488, 485 Annotation: Subtype: Link Rect: 304, 451, 342, 462 Annotation: Subtype: Link Rect: 217, 429, 254, 439 Annotation: Subtype: Link Rect: 261, 429, 297, 439 Annotation: Subtype: Link Rect: 306, 394, 344, 404 Annotation: Subtype: Link Rect: 446, 370, 483, 381 Annotation: Subtype: Link Rect: 490, 370, 526, 381 Annotation: Subtype: Link Rect: 393, 347, 431, 358 Annotation: Subtype: Link Rect: 437, 347, 473, 358 Annotation: Subtype: Link Rect: 393, 324, 430, 335 Annotation: Subtype: Link Rect: 436, 324, 472, 335 Annotation: Subtype: Link Rect: 140, 302, 178, 312 Annotation: Subtype: Link Rect: 184, 302, 220, 312 Annotation: Subtype: Link Rect: 244, 267, 282, 278 Annotation: Subtype: Link Rect: 288, 267, 324, 278 Annotation: Subtype: Link Rect: 104, 233, 142, 243 Annotation: Subtype: Link Rect: 477, 209, 515, 220 Annotation: Subtype: Link Rect: 521, 209, 557, 220 Annotation: Subtype: Link Rect: 59, 175, 97, 186 Annotation: Subtype: Link Rect: 140, 141, 178, 151 Annotation: Subtype: Link Rect: 126, 118, 164, 128 Annotation: Subtype: Link Rect: 100, 60, 137, 70 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 414, 721, 452, 732 Annotation: Subtype: Link Rect: 475, 698, 512, 709 Annotation: Subtype: Link Rect: 519, 698, 555, 709 Annotation: Subtype: Link Rect: 520, 675, 557, 686 Annotation: Subtype: Link Rect: 59, 664, 95, 674 Annotation: Subtype: Link Rect: 238, 629, 275, 640 Annotation: Subtype: Link Rect: 281, 629, 317, 640 Annotation: Subtype: Link Rect: 159, 595, 196, 605 Annotation: Subtype: Link Rect: 388, 548, 425, 559 Annotation: Subtype: Link Rect: 95, 515, 133, 525 Annotation: Subtype: Link Rect: 59, 492, 97, 502 Annotation: Subtype: Link Rect: 503, 468, 541, 479 Annotation: Subtype: Link Rect: 95, 446, 133, 456 Annotation: Subtype: Link Rect: 182, 422, 219, 433 Annotation: Subtype: Link Rect: 374, 410, 412, 421 Annotation: Subtype: Link Rect: 520, 399, 557, 410 Annotation: Subtype: Link Rect: 452, 376, 489, 387 Annotation: Subtype: Link Rect: 495, 376, 531, 387 Annotation: Subtype: Link Rect: 190, 354, 228, 364 Annotation: Subtype: Link Rect: 234, 354, 270, 364 Annotation: Subtype: Link Rect: 329, 330, 366, 341 Annotation: Subtype: Link Rect: 127, 308, 164, 318 Annotation: Subtype: Link Rect: 378, 272, 415, 283 Annotation: Subtype: Link Rect: 519, 249, 557, 260 Annotation: Subtype: Link Rect: 59, 238, 95, 249 Annotation: Subtype: Link Rect: 258, 215, 294, 226 Annotation: Subtype: Link Rect: 104, 181, 142, 191 Annotation: Subtype: Link Rect: 476, 146, 514, 157 Annotation: Subtype: Link Rect: 59, 112, 97, 122 Annotation: Subtype: Link Rect: 276, 77, 314, 88 Annotation: Subtype: Link Rect: 320, 77, 356, 88 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 509, 709, 547, 720 Annotation: Subtype: Link Rect: 351, 675, 388, 686 Annotation: Subtype: Link Rect: 159, 640, 196, 651 Checksum: 485ad127 Type: CRC32 Checksum: b6637a239ce95d3ab1bd3c89fac2eef8 Type: MD5 Checksum: bc2334b54ceed4e717835beef1527a8858e942b4 Type: SHA-1