Volkhard Rippe

The expression pattern of the Hmgic gene during development

The technique of RNA in situ hybridization to mouse embryo sections from different developmental stages was used to perform a detailed analysis of the expression pattern of the gene for the architectural chromatin factor Hmgic. At early stages of fetal development (day 9.5 post conceptionem), Hmgic is expressed at a high rate throughout the whole embryo. In the second half of development, the pattern of expression becomes more restricted. Expression is found in mesenchymal derivatives, which differentiate into cartilage or muscle, in epithelial cell layers of the lung, pancreas, submandibular gland, and vibrissae, and in some special parts of the central nervous system. The expression pattern of Hmgic was compared with the previously reported studies of Hmgiy gene expression, another member of the Hmgic protein family, and with the expression of histone H4, Hist4, which is representative of cellular proliferation stages. In some tissues the pattern of expression for both factors coincides, but in others the expression is different. Hmgic expression correlates throughout fetal development with high proliferative activity. In contrast, Hmgiy is expressed also in tissues with no proliferative activity, such as the cortical plate of the telencephalon and the spinal cord at late gestational stages. Genes Chromosomes Cancer 23:350–357, 1998.

Expression of HMGI-C, a member of the high mobility group protein family, in a subset of breast cancers: Relationship to histologic grade

The high‐mobility‐group (HMG) protein gene HMGI‐C is apparently involved in the genesis of a variety of benign human solid tumors with rearrangements of chromosomal region 12q14‐15 affecting the HMGI‐C gene. So far, no expression of HMGI‐C has been found in adult tissues, and no data are available on the expression of HMGI‐C in primary human malignant tumors of epithelial origin. Therefore, we analysed the HMGI‐C expression patterns in 44 breast cancer samples and 13 samples of nonmalignant adjacent tissue by hemi‐nested reverse transcriptase–polymerase chain reaction for HMGI‐C expression. There was no detectable expression of HMGI‐C in any nonmalignant adjacent breast tissues analyzed. In contrast, we found expression in 20 of 44 breast cancer samples investigated. In invasive ductal tumors, expression was noted predominantly in tumors with high histologic grade: 17 of 21 breast cancer samples with histologic grade 3 but only three of 16 samples with histologic grades 1 or 2 showed expression of HMGI‐C. In addition, all seven lobular breast cancer samples tested did not express HMGI‐C. From these results, we concluded that HMGI‐ C expression may be of pathogenetic or prognostic importance in breast cancer. Mol. Carcinog. 19:153–156, 1997.

The Two Stem Cell MicroRNA Gene Clusters C19MC and miR-371-3 Are Activated by Specific Chromosomal Rearrangements in a Subgroup of Thyroid Adenomas

Thyroid adenomas are common benign human tumors with a high prevalence of about 5% of the adult population even in iodine sufficient areas. Rearrangements of chromosomal band 19q13.4 represent a frequent clonal cytogenetic deviation in these tumors making them the most frequent non-random chromosomal translocations in human epithelial tumors at all. Two microRNA (miRNA) gene clusters i.e. C19MC and miR-371-3 are located in close proximity to the breakpoint region of these chromosomal rearrangements and have been checked for a possible up-regulation due to the genomic alteration. In 4/5 cell lines established from thyroid adenomas with 19q13.4 rearrangements and 5/5 primary adenomas with that type of rearrangement both the C19MC and miR-371-3 cluster were found to be significantly overexpressed compared to controls lacking that particular chromosome abnormality. In the remaining cell line qRT-PCR revealed overexpression of members of the miR-371-3 cluster only which might be due to a deletion accompanying the chromosomal rearrangement in that case. In depth molecular characterization of the breakpoint in a cell line from one adenoma of this type reveals the existence of large Pol-II mRNA fragments as the most likely source of up-regulation of the C19MC cluster. The up-regulation of the clusters is likely to be causally associated with the pathogenesis of the corresponding tumors. Of note, the expression of miRNAs miR-520c and miR-373 is known to characterize stem cells and in terms of molecular oncology has been implicated in invasive growth of epithelial cells in vitro and in vivo thus allowing to delineate a distinct molecular subtype of thyroid adenomas. Besides thyroid adenomas rearrangements of 19q13.4 are frequently found in other human neoplasias as well, suggesting that activation of both clusters might be a more general phenomenon in human neoplasias.

A characteristic sequence of trisomies starting with trisomy 7 in benign thyroid tumors

The cytogenetic results from a series of 113 thyroid hyperplasias and adenomas are reported; 15 showed clonal karyotypic alterations. In addition to a group showing translocations involving 19q13, another subset of lesions characterized by polysomies can be found. Based on our own cases belonging to this subset and a review of the cases reported in the literature, we conclude that the characteristic feature of this group is a sequence that always starts with trisomy 7, but that sometimes even leads to chromosome numbers in the hypertriploid range. This subset of thyroid tumors may be an example of a more common genetic pathway in human solid tumors.

Identification of a gene rearranged by 2p21 aberrations in thyroid adenomas.

Thyroid adenomas belong to the cytogenetically best investigated human epithelial tumors. Cytogenetic studies of about 450 benign lesions allow one to distinguish between different cytogenetic subgroups. Two chromosomal regions, that is, 19q13 and 2p21, are frequently rearranged in these tumors. Although 2p21 aberrations only account for about 10% of the benign thyroid tumors with clonal cytogenetic deviations, 2p21 rearrangements belong to the most common cytogenetic rearrangements in epithelial tumors due to the high frequency of these benign lesions. The 2p21 breakpoint region recently has been delineated to a region of 450 kbp, but the gene affected by the cytogenetic rearrangements still has escaped detection. Positional cloning and 3′ RACE–PCR allowed us to clone that gene which we will refer to as thyroid adenoma associated (THADA) gene. In cells from two thyroid adenomas characterized by translocations t(2;20;3) (p21;q11.2;p25) and t(2;7)(p21;p15), respectively, we performed 3′-RACE–PCRs and found two fusions of THADA with a sequence derived from chromosome band 3p25 or with a sequence derived from chromosome band 7p15. The THADA gene spans roughly 365 kbp and, based on preliminary results, encodes a death receptor-interacting protein.

A KRAB zinc finger protein gene is the potential target of 19q13 translocation in benign thyroid tumors

In an attempt to identify the target gene of specific translocations involving chromosomal band 19q13 in benign follicular thyroid tumors, we have used two cell lines derived from benign thyroid tumors showing translocations with 19q13 breakpoints for fluorescence in situ hybridization mapping studies with cosmid and PAC clones located in a 400‐kbp region. The breakpoints of the chromosome 19 abnormalities mapped within a 140‐kb segment covered by a single PAC clone. Sequencing of part of this PAC clone allowed us to establish the cDNA sequence and the genomic structure of a candidate gene located in close vicinity to the breakpoints. The gene that we tentatively refer to as RITA (rearranged in thyroid adenomas) belongs to the KRAB zinc finger protein coding genes. From our results we have concluded that in the two cell lines investigated the breaks have occurred either within the 5′ untranslated region of RITA or in its close 5′ vicinity. By Northern blot analyses two transcripts of about 4.7 kbp and 5 kbp were detected in normal thyroid tissue as well as in other normal tissues tested. An additional 2.1‐kbp transcript was found only in testicular tissue. In contrast to all normal tissues, both cell lines with 19q aberrations expressed larger transcripts of approximately 5.5 kbp and 6.2 kbp. From the close vicinity to the breakpoint region, the expression patterns of the gene, and its type, we consider RITA a strong candidate target gene of the specific 19q aberrations in benign thyroid tumors. Genes Chromosomes Cancer 26:229–236, 1999.

Chromosomal rearrangements leading to abnormal splicing within intron 4 of HMGIC

Fusion of the high‐mobility group protein gene HMGIC to other genes due to chromosomal rearrangements occurs in a variety of human benign tumors. In contrast to genes clearly derived from other chromosomes, some of the ectopic sequences fused to HMGIC have been assigned to chromosome 12 by CASH (chromosome assignment using somatic cell hybrids) analyses and thus can be assumed either to result from alternative splicing or to represent true ectopic sequences derived from other genes on chromosome 12. In an attempt to identify the ectopic sequences fused to this exon, we have sequenced the entire intron 4. Four of seven ectopic sequences previously described to be fused to exon 4 of HMGIC in different tumors were found to be located within intron 4 of the gene and thus are due to abnormal splicing. As for a mechanism explaining this observation, it can be suggested that breakpoints of chromosomal aberrations not directly disrupting HMGIC may induce small genomic alterations in their vicinity and thus facilitate abnormal splicing. The latter mechanism may underlie the development of part of the neoplasms characterized by 12q14–15 rearrangements.

Breakpoints of 19q13 translocations of benign thyroid tumors map within a 400 kilobase region.

Structural rearrangements involving the long arm of chromosome 19 characterize a cytogenetic subgroup of benign thyroid tumors. To localize the breakpoint of the 19q13 aberrations, we have established three cell lines derived from benign thyroid tumors showing translocations in this region. We have used these cell lines and four additional primary tumors with 19q13 abnormalities for fluorescence in situ hybridization (FISH) mapping studies with ten cosmid clones located between the molecular markers POLD1 and TNNT1. The breakpoints of all chromosome 19 abnormalities mapped within a 400 kb region. Genes Chromosomes Cancer 20:201–203, 1997.

On the prevalence of the PAX8-PPARG fusion resulting from the chromosomal translocation t(2;3)(q13;p25) in adenomas of the thyroid.

The chromosomal translocation t(2;3)(q13;p25) characterizes a subgroup of tumors originating from the thyroid follicular epithelium and was initially discovered in a few cases of adenomas. Later, a fusion of the genes PAX8 and PPARG resulting from this translocation was frequently observed in follicular carcinomas and considered as a marker of follicular thyroid cancer. According to subsequent studies, however, this rearrangement is not confined to carcinomas but also occurs in adenomas, with considerably varying frequencies. Only five cases of thyroid adenomas with this translocation detected by conventional cytogenetics have been documented. In contrast, studies using reverse-transcription polymerase chain reaction (RT-PCR) detected fusion transcripts resulting from that translocation in an average of 8.2% of adenomas. The aim of this study was to determine the frequency of the PAX8-PPARG fusion in follicular adenomas and to use the HMGA2 mRNA level of such tumors as an indicator of malignancy. In cytogenetic studies of 192 follicular adenomas, the t(2;3)(q13;p25) has been identified in only two cases described herein. Histopathology revealed no evidence of malignancy in either case, and, concordantly, HMGA2 mRNA levels were not elevated. In summary, the fusion is a rare event in follicular adenomas and its prevalence may be overestimated in many RT-PCR-based studies.

Activation of the two microRNA clusters C19MC and miR-371-3 does not play prominent role in thyroid cancer.

Chromosomal rearrangements of band 19q13.4 are frequent cytogenetic alterations in benign thyroid adenomas. Apparently, these alterations lead to the upregulation of genes encoding microRNAs of two clusters mapping to the breakpoint region, i.e. miR-371-3 and C19MC. Since members of both clusters have been associated with neoplastic growth in other tumor entities the question arises whether or not their upregulation predisposes to malignant transformation of follicular cells of the thyroid. To address this question we have quantified the expression of miR-372 and miR-520c-3p in samples of 114 thyroid cancers including eight anaplastic thyroid carcinomas, 25 follicular thyroid carcinomas, 78 papillary thyroid carcinomas (including 13 follicular variants thereof), two medullary thyroid carcinomas and one oncocytic thyroid carcinoma. Additionally, we quantified miR-371a-3p and miR-519a-3p in selected samples. While in neither of the cases miR-520c-3p and miR-519a-3p were found to be upregulated, one papillary and one anaplastic thyroid carcinoma, respectively, showed upregulation of miR-372 and miR-371a-3p. However, in these cases fluorescence in situ hybridization did not reveal rearrangements of the common breakpoint region as affected in adenomas. Thus, these rearrangements do apparently not play a major role as first steps in malignant transformation of the thyroid epithelium. Moreover, there is no evidence that 19q13.4 rearrangements characterize a subgroup of thyroid adenomas associated with a higher risk to undergo malignant transformation. Vice versa, the mechanisms by which 19q13.4 rearrangements contribute to benign tumorigenesis in the thyroid remain to be elucidated.