Reprinted with permission from the American Journal of Pathology, Bethesda, MD.
As published in American Journal
of Pathology Vol. 150, No. 3, pp. 911-917, 1997
Amy J. Williams, W. Lee Powell,
Tucker Collins, and Cynthia C. Morton
From the Departments of Pathology, Brigham and Women's Hospital and Harvard
Medical School, Boston, Massachusetts
Chromosomal rearrangements
involving 6p2l have been observed in uterine leiomyomata and a variety of other
benign tumors. The gene for HMGI(Y), a member of the high-mobility group (HMG)
family of proteins, has been localized to 6p2l. To determine whether
rearrangements observed in this area alter HMGI(Y) expression, we analyzed
HMGI(Y) DNA-binding activity in protein extracts from uterine leiomyoma and
normal myometrium tissues. This report describes a uterine leiomyoma specimen
with an inv(6)(p2lql5). A genomic PI clone that contains the HMGI(Y) region of
chromosome 6 is found to span the inversion breakpoint by fluorescent in situ
hybridization of metaphase chromosomes. Expression of HMGI(Y)protein in this
leiomyoma specimen is increased dramatically as compared with the matching
normal myometrial tissue. Elevated HMGI(Y) expression was also found in 8 of 16
leiomyomas without cytogenetically detectable chromosome 6P21 aberrations but
not in any of the 9 matching myometrial tissues. Analysis of the genetic events
involved in the pathobiolgy of these benign tumors will provide a basis for
understanding the process of improper cellular growth and might be important in
deciphering the multistep pathway of tumorigenesis. (Am J Pathol 1997,
150:911-918)
Table 1. Karyotype of Uterine Leiomyomata and
Semiquantitative Analysis of HMGI(Y) Binding Activity
UL, uterine leiomyoma; M, myometrium.
Each UL specimen was taken from an independent tumor in cases in which a patient
had multiple fibroids. ND, not determined; -, no detectable HMGI(Y) binding.
Quantitative analysis of positive HMGI(Y) binding activity was determined by
comparing the relative intensities of DNA-protein complexes from EMSA
autoradiographs and by comparing the weights (in mg) of shifted bands when cut
out from a copy of the autoradiograph.
*Karyotypes have been previously reported. 34
+Karyotypes have been previously reported. 8
#Karyotype has been previously reported.35
Figure 1. In situ hybridization with a PI genomic clone reveals that a 6p21 breakpoint in a uterine leiomyoma is near the HMGI(Y) gene. A: FISH analysis of leiomyoma ST93-397 metaphase chromosomes with PI clone 6995. Metaphase spread from ST93-397, (46,XY, inv6(p2lql5), inv9), hybridized with digoxigenin-labeled PI clone 6995 and detected with rhodamine-labeled anti-digoxigenin. Hybridization signals on the normal chromosome 6 are indicated by a red arrow, and signals on the inv6(p2lql5) chromosome are indicated by a green arrow. The inset is a GTG-banded partial karyotype of ST93-397 demonstrating the inv6(p2lql5). B: Partial restriction map and schematic representation of PI clone 6995 showing the location of the HMGI(Y) gene (exon-intron boundaries within the gene are not indicated). The arrow in front of the gene indicates the transcriptional start site. The arrows at the ends of the PI clone 6995 are meant to indicate that the clone extends beyond what is shown in the figure.
Figure 2.HMGI(Y) DNA-binding activity is increased in a uterine leiomyoma with an inv6(p2lql5). A: Sequence of oligonucleotides PRDII X 2 and MPRDII X 2 used in EMSA analysis. The duplicated NF-kB site (PRDII)16 from the human IFN-B promoter has been underlined. Bolded nucleotides show the HMGI(I) binding site and asterisks above the mPRDII X 2 sequence indicate those nucleotides that were changed to mutate the binding site. B: EMSA analysis of protein extract from leiomyoma ST93-397. Lanes 1 to 4 contain P-labeled PRDII X 2 oligonucleotide probe mixed with no extract (lane 1), 4 ug of total protein extract from normal myometrium tissue ST93-398 (lane 2), 4 ug of total protein extract from leiomyoma tissue ST93-397 (lane 3), and recombinant p50 (lane 4). Lanes 5 to 8 contain P-labeled MPRDII X 2 mixed with no extract (lane 5 ), 4 ug of total protein extract from normal myometrium ST93-398 (lane 6), 4 ug of total protein extract from leiomyoma ST93-397 (lane 7), and recombinant p50 (lane 8). Recombinant p50 homodimer (a form of NF-kB) was used to demonstrate the integrity of both wild-type and mutated PRDII X 2 oligonucleotides. Bound complexes are indicated by an arrow to the right of the figure. FREE indicates the location of unbound probe.
Figure 3.EMSA analysis of protein extracts from 16 uterine leiomyomas and 10 matched myometrial specimens. Lanes contain P-labeled PRDII X 2 oligonucleotide probe mixed with no protein extract (lane 1) or 13 g of total protein extract from leiomyoma or myometrium (lanes 2 to 26). Leiomyoma specimen numbers are followed by the letter L whereas myometrium specimen numbers are follotwd by the letter M. Brackets above the specimen number indicate that the tissue samples were obtained from the same patient. The arrow to the right of tbe figure identifies DNA-protein complexes that can be abolished by substituting the oligonucleotide probe mPPDIIX 2 in the EMSA reaction. FREE indicates tbe position of unbound probe.