6. 1 Summary
Feline fibrosarcomas are the most common skin tumors of cats. Despite this high frequency and the publication of different hypotheses for their pathogenesis by several authors, the alterations accompanying the development of this tumor are still not completely understood.
We studied the feline major satellite DNA (FA-SAT) hybridization pattern by FISH in four fibrosarcoma cell lines and one normal embryonic fibroblastic cell line as well as in 30 fibrosarcomas from 28 cats. Of the latter, tumor tissue biopsies and biopsies of non-affected skin of the same patient were analyzed.
The hybridization pattern of the FA-SAT in the normal fibroblastic cell line differed significantly from that observed in all fibrosarcoma cell lines. The same observation was made for tumor tissues and fibroblasts from non-affected skin in paraffin-embedded material. These differences observed in vitro and in vivo suggest that alterations of the FA-SAT may be associated with chromosomal instability in the feline fibrosarcoma.
Additionally, our FISH results show the usability of the designed method in differentiating tumor cells from normal skin fibroblasts in feline fibrosarcoma.
Index of figures
FIGURE 1:MAJOR FELINE SATELLITE DNA REPORTED BY FANNIG (1987) ... 13 FIGURE 2:RESTRICTION MAP AND CLONING SITE AND EXPRESSION REGION OF THE
,,PSTBLUE-1”VECTOR (NOVAGEN,MERCK,DARMSTADT) ... 25 FIGURE 3:2% GEL ELECTROPHORESIS IN WHICH THE PCR PRODUCTS OF THE PRIMER TEST
ARE SHOWN... 43 FIGURE 4:2% GEL ELECTROPHORESIS OF THE PCR REACTION WITH SPECIFIC
OLIGONUCLEOTIDE PRIMERS FOR THE FA-SATDNA ON BACTERIAL COLONIES... 44 FIGURE 5:INSERTED DNA SEQUENCE OF THE PAA16 CLONE WITH 95% HOMOLOGY TO
PUBLISHED SATELLITE DNA FAMILY (FA-SAT) IN THE DOMESTIC CAT... 45 FIGURE 6:CONTROL CELL LINE IN WHICH LARGE SIGNALS AND SMALL SIGNALS CAN BE
APPRECIATED... 46 FIGURE 7:QUANTITATIVE DISTRIBUTION OF THE NUMBER OF LARGE SIGNALS PER TUMOR
CELL IN THE FELINE FIBROSARCOMA CELL LINE FSI... 47 FIGURE 8:QUANTITATIVE DISTRIBUTION OF THE NUMBER OF LARGE SIGNALS PER TUMOR
CELL IN THE FELINE FIBROSARCOMA CELL LINE FSII ... 48 FIGURE 9:QUANTITATIVE DISTRIBUTION OF THE NUMBER OF LARGE SIGNALS PER TUMOR
CELL IN THE FELINE FIBROSARCOMA CELL LINE FSIII ... 49 FIGURE 10:QUANTITATIVE DISTRIBUTION OF THE NUMBER OF LARGE SIGNALS PER TUMOR
CELL IN THE FELINE FIBROSARCOMA CELL LINE FSIV... 50 FIGURE 11:QUANTITATIVE DISTRIBUTION OF THE NUMBER OF LARGE SIGNALS PER CELL IN THE CONTROL CELL LINE... 51 FIGURE 12:BOX-AND-WHISKER-PLOTS OF THE TOTAL NUMBER OF LARGE SIGNALS PER
CELL OF THE STUDIED CELL LINES... 52 FIGURE 13:QUANTITATIVE DISTRIBUTION OF THE NUMBER OF SMALL SIGNALS PER TUMOR CELL IN THE FELINE FIBROSARCOMA CELL LINE FSΙ... 53 FIGURE 14:QUANTITATIVE DISTRIBUTION OF THE NUMBER OF SMALL SIGNALS PER TUMOR CELL IN THE FELINE FIBROSARCOMA CELL LINE FSII ... 54 FIGURE 15:QUANTITATIVE DISTRIBUTION OF THE NUMBER OF SMALL SIGNALS PER TUMOR CELL IN THE FELINE FIBROSARCOMA CELL LINE FSIII ... 55 FIGURE 16:QUANTITATIVE DISTRIBUTION OF THE NUMBER OF SMALL SIGNALS PER TUMOR CELL IN THE FELINE FIBROSARCOMA CELL LINE FSVI... 56
FIGURE 17:QUANTITATIVE DISTRIBUTION OF THE NUMBER OF SMALL SIGNALS PER CELL IN THE FELINE SKIN FIBROBLASTS... 57 FIGURE 18:BOX-AND-WHISKER-PLOTS OF THE TOTAL NUMBER OF SMALL SIGNALS PER
CELL OF THE STUDIED CELL LINES... 58 FIGURE 19:HYBRIDIZATION PATTERN OF THE FELINE MAJOR SATELLITE DNA(FA-SAT)
IN CELL LINES... 59 FIGURE 20:BOX-AND-WHISKER-PLOT OF THE TOTAL NUMBER OF SIGNALS PER CELL IN
TUMOR TISSUE AND DERMAL FIBROBLASTS OF NORMAL SKIN OF 28 PATIENTS... 61 FIGURE 21:HYBRIDIZATION PATTERN OBSERVED IN TISSUE SECTIONS OF FIBROBLASTS OF
NON-AFFECTED SKIN, AND FELINE FIBROSARCOMA TUMOR CELLS... 62 FIGURE 22:BOX-AND-WHISKER-PLOT OF THE NUMBER OF SIGNALS PER CELL OF TUMOR
CELLS OF THE FIRST TUMOR (T6035/04) AND ITS RECURRENCE (T8904/04) AS WELL AS THEIR CORRESPONDENT SKIN BIOPSIES... 63 FIGURE 23:BOX-AND-WHISKER-PLOT OF THE NUMBER OF SIGNALS PER CELL OF TUMOR
CELLS OF THE FIRST TUMOR (T6125/05) AND ITS RECURRENCE (T8535/06) AS WELL AS OF THEIR CORRESPONDENT SKIN BIOPSIES... 64 FIGURE 24:ESTIMATED NORMAL DISTRIBUTIONS OF THE MEAN NUMBERS OF SIGNALS PER
CELL IN TUMOR TISSUE AND FIBROBLASTS FROM NON-AFFECTED SKIN WITH A SAMPLE SIZE OF 100 CELLS (TUMOR AND NON-AFFECTED FIBROBLASTS) TAKEN FROM 28 CATS
... 65 FIGURE 25:ESTIMATED NORMAL DISTRIBUTIONS OF THE MEAN NUMBERS OF SIGNALS PER
CELL IN TUMOR TISSUE AND FIBROBLASTS FROM NON-AFFECTED SKIN WITH A SAMPLE SIZE OF 50 CELLS (TUMOR AND NON-AFFECTED FIBROBLASTS) TAKEN FROM 28 CATS
... 65 FIGURE 26:ESTIMATED NORMAL DISTRIBUTIONS OF THE MEAN NUMBERS OF SIGNALS PER
CELL IN TUMOR TISSUE AND FIBROBLASTS FROM NON-AFFECTED SKIN WITH A SAMPLE SIZE OF 25 CELLS (TUMOR AND NON-AFFECTED FIBROBLASTS) TAKEN FROM 28 CATS
... 66 FIGURE 27:ROC-PLOT ASSUMING THE NORMAL DISTRIBUTION MODEL FOR THREE CELL
Index of tables
TABLE 1:SEQUENCE AND ORIENTATION OF THE PRIMERS FOR THE FA-SATDNA... 21 TABLE 2:PCR REACTION COMPOSITION FOR THE BIOTHERM TMTAQ-POLYMERASE
(NATUTEC,FRANKFURT) ... 22 TABLE 3:PRC REACTION CONDITIONS FOR THE BIOTHERM TMTAQ-POLYMERASE
(NATUTEC,FRANKFURT) ... 22 TABLE 4:LIGATION REACTION MIXTURE... 27 TABLE 5:PCR MASTER MIX COMPOSITION FOR BACTERIAL COLONIES ... 29 TABLE 6:PCR REACTION CONDITIONS FOR BACTERIAL COLONIES... 29 TABLE 7:CONDITIONS FOR THE DIGESTION REACTION OF FIVE SELECTED BACTERIAL
COLONIES... 31 TABLE 8:RESTRICTION DIGESTION WITH ECO57MI ... 32 TABLE 9:REAGENTS FOR THE T7RNA POLYMERASE TRANSCRIPTION... 33 TABLE 10:CONCENTRATION OF THE ISOLATED DNA WITH THE GENTRA PUREGENE® KIT
(GENTRA-SYSTEMS,MINESOTA,USA) ... 42 TABLE 11:STATISTICALLY SIGNIFICANT DIFFERENCES BETWEEN THE DIFFERENT FELINE
FIBROSARCOMA CELL LINES FOUND WITH A POST-HOC-TEST (NEMENYI) ... 58