Expression and interdependencies of pluripotency factors LIN28, OCT3/4, NANOG and SOX2 in human testicular germ cells and tumours of the testis.

OCT3/4, NANOG, SOX2 and, most recently, LIN28 have been identified as key regulators of pluripotency in mammalian embryonic and induced stem cells, and are proven to be crucial for generation of the mouse germ-cell lineage. These factors are a hallmark of certain histological types of germ-cell tumours (GCTs). Here, we report novel information on the temporal and spatial expression pattern of LIN28 during normal human male germ-cell development as well as various types of GCTs. To investigate LIN28 expression, immunohistochemical analyses and quantitative proximity ligation assay-based TaqMan protein assays were applied on snap-frozen and formalin-fixed, paraffin-embedded samples as well as representative cell lines. LIN28 was found in primordial germ cells, gonocytes and pre-spermatogonia, in contrast to OCT3/4 and NANOG, which were found only in the first two stages. LIN28 was also found in all precursor lesions (carcinoma in situ and gonadoblastoma) of type II GCTs, as well as the invasive components seminoma and the non-seminomatous elements embryonal carcinoma and yolk sac tumour. Choriocarcinoma showed a heterogeneous pattern, while teratomas and spermatocytic seminomas (type III GCTs) were negative. This expression pattern suggests that LIN28 is associated with malignant behaviour of type II GCTs. Cell line experiments involving siRNA knockdown of LIN28, OCT3/4 and SOX2 showed that LIN28 plays a role in the maintenance of the undifferentiated state of both seminoma and embryonal carcinoma, closely linked to, and likely upstream of OCT3/4 and NANOG. In conclusion, LIN28 regulates the differentiation status of seminoma and embryonal carcinoma and is likely to play a related role in normal human germ-cell development.

Biallelic expression of the H19 and IGF2 genes in human testicular germ cell tumors.

BACKGROUND: Genomic imprinting, resulting in the nonequivalence of expression of homologue genes depending on their parental origin, is an important determinant of the developmental potential of embryonic cells. The expression of two genes, one termed H19 and the other IGF2, has been found to be necessary for proper embryonal development in humans. Both the murine and human H19 and IGF2 genes are normally characterized by monoallelic expression. PURPOSE: Because testicular germ cell tumors of adults originate from an early germ cell and, to a certain extent, mimic normal embryonal development, we investigated the patterns of allelic expression of the H19 and IGF2 genes in these tumors to determine if genomic imprinting, or a disturbance of it, is involved in their pathogenesis. METHODS: Specimens of normal tissue and tumor tissue were obtained from 20 patients with testicular germ cell tumors; 10 of the patients had seminomas and 10 had nonseminomatous germ cell tumors. To determine if there was heterozygosity of the Alu I and Apa I restriction site polymorphism in the H19 and IGF2 genes, respectively, DNA was isolated from cells of the peripheral blood of these patients, then subjected to polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and electrophoresis in agarose gels. If heterozygosity was determined, a similar analysis was performed on complementary DNA (cDNA) obtained from matched tumor RNA by reverse transcription and subsequent PCR amplification (RT-PCR). If monoallelic expression was found, matched tumor DNA was studied for possible deletions. RESULTS: Tumor samples from 14 of 20 and 11 of 20 patients were informative for allelic expression patterns of the H19 and IGF2 genes, respectively. Analysis of the products of RT-PCR showed biallelic expression of the H19 gene in 12 testicular germ cell tumors (patients numbered 6, 8-13, 15, 16, and 18-20) and of the IGF2 gene in 10 testicular germ cell tumors (patients numbered 1, 3, 6, 8-13, and 15-20). The three remaining tumors (patients numbered 2, 4, and 5) had lost the nonexpressed allele. CONCLUSIONS: In contrast to normally developing embryos, testicular germ cell tumors show a consistent expression of both parental alleles of the H19 and IGF2 genes. IMPLICATIONS: Testicular germ cell tumors of adults may develop from precursor cells in which the imprinting has been either erased or subjected to a consistent relaxation of its effect.

Overrepresentation of the short arm of chromosome 12 is related to invasive growth of human testicular seminomas and nonseminomas.

Overrepresentation of 12p-sequences, mostly due to isochromosome formation, is the only consistent chromosomal alteration found in invasive testicular germ cell tumors of adolescents and young adults (TGCTs), both seminomas and the various histological elements of nonseminomas. The biological role of extra 12p in the pathogenesis of this cancer is unclear, and it is also unknown so far, whether it is an early event, i.e., already present in carcinoma in situ, or related to invasive growth. Using comparative genomic hybridization (CGH) with DOP-PCR amplified DNA isolated from micro-dissected tumor cells, and double fluorescent in situ hybridization (FISH) on frozen tissue sections, we investigated the presence of overrepresentation of 12p sequences in different development stages of four seminomas and seven nonseminomas, in total 17 invasive components, in addition to the carcinoma in situ of each. CGH demonstrated relative gain of 12p-sequences in all invasive components except one, confirmed by FISH in most samples. In contrast, no gain was found in the carcinoma in situ samples by any of the methods. These findings show that overrepresentation of 12p is not an early event in the development of TGCTs, but relates to invasive growth.

Unique expression patterns of H19 in human testicular cancers of different etiology.

The expression pattern of the imprinted human H19 gene was investigated in testicular cancers of different etiology, as well as in normal testicular parenchyma, parenchyma without germ cells, and adjacent to testicular germ cell tumors of adolescents and adults (TGCTs), using RNase protection analysis, mRNA in situ hybridization and reverse-transcription polymerase chain reaction. While different total expression levels were detected in spermatocytic seminomas, lymphomas, a Sertoli cell tumor and Leydig cell tumors, none showed a disturbance of monoallelic expression. Strikingly, the majority of invasive TGCTs revealed expression of both parental alleles. The total level of expression highly correlated with differentiation lineage and stage of maturation, similar to that as reported during early normal embryogenesis. Biallelic expression could also be determined specifically in testis parenchyma containing the preinvasive lesion of this cancer. We therefore conclude that within the adult testis, biallelic H19 expression is specific for TGCTs, and that the level of expression is dependent on differentiation lineage and maturation stage. This is in agreement with the proposed primordial germ cell-origin of this cancer, and might be related to retention of embryonic characteristics in TGCTs. In addition, our data argue against H19 being a tumor suppressor gene.

Human growth-differentiation factor 3 (hGDF3): developmental regulation in human teratocarcinoma cell lines and expression in primary testicular germ cell tumours.

We describe the cloning and initial characterization of a novel cDNA from human embryonal carcinoma (EC) cells. This cDNA, which we named human growth differentiation factor 3 (hGDF3), encodes the homologue of mouse GDF3, a TGFbeta superfamily member belonging to the Growth/Differentiation Factors. We have analysed the expression of hGDF3 in human embryonal carcinoma cell lines and in primary testicular germ cell tumours of adolescents and adults (TGCTs). Expression of hGDF3 in human EC cell lines is stem cell-specific, is down-regulated upon RA-mediated differentiation and is increased upon culture of the cells in the presence of activin A. In TGCTs, hGDF3 expression is low in seminomas, while expression in non-seminomas is readily detectable and appears to be associated with the EC and yolk sac components in the tumours. We have also mapped the hGDF3 locus to the short arm of human chromosome 12, a region consistently overrepresented in human testicular germ cell tumours. Thus, hGDF3 represents an embryonal carcinoma stem cell-associated marker both in vitro and in vivo.

Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11.2-p12.1.

Gain of 12p material is invariably associated with testicular germ cell tumors (TGCTs) of adolescents and adults, most usually as an isochromosome 12p. We analyzed TGCTs with i(12p) using a global approach to expression profiling targeting chromosomes (comparative expressed sequence hybridization, CESH). This indicated overexpression of genes from 12p11.2-p12.1 relative to testis tissue and fibroblasts. The nonseminoma subtype showed higher levels of expression than seminomas. Notably, 12p11.2-p12.1 is amplified in about 10% of TGCTs and CESH analysis of such amplicon cases showed high levels of overexpression from this region. Microarray analysis, including cDNA clones representing most UniGene clusters from 12p11.2-p12.1, was applied to DNA and RNA from 5 TGCTs with amplification of 12p11.2-p12.1 and seven TGCTs with gain of the entire short arm of chromosome 12. Expression profiles were consistent with the CESH data and overexpression of EST595078, MRPS35 and LDHB at 12p11.2-p12.1 was detected in most TGCTs. High-level overexpression of BCAT1 was specific to nonseminomas and overexpression of genes such as CMAS, EKI1, KRAS2, SURB7 and various ESTs correlated with their amplification. Genes such as CCND2, GLU3, LRP6 and HPH1 at 12p13 were also overexpressed. The overexpressed sequences identified, particularly those in the region amplified, represent candidate genes for involvement in TGCT development.

Phenotyping of acute myelocytic leukemia (AML) progenitors: an approach for tracing minimal numbers of AML cells among normal bone marrow.

Previous studies have shown that the phenotypes of progenitors of human AML (AML-CFU) are variable, reflecting arrests at different stages of maturation. We were interested to seek discrepancies between the surface properties of AML precursors and normal bone marrow colony formers in order to detect minimal numbers of AML cells among normal bone marrow cells in remission bone marrow. Therefore, we selected two surface markers, the MoAb CD34, reactive with blast cells, and Vim-2, a surface marker reactive with mature myeloid cells, and determined the antigen density of these markers (relative fluorescence intensity using fluorescence-activated cell sorting) for normal marrow and AML progenitors. While these markers defined an identical phenotype (CD34++/Vim-2-/+) for a broad spectrum of normal progenitors, i.e., CFU-GEMM, BFU-e, day 15 CFU-GM, and day 7 CFU-GM, referred to as the "normal" progenitor phenotype, AML progenitors frequently exhibited different phenotypes. In 12 of 20 cases the phenotypes of the majority of AML progenitors were discrepant from the normal surface profile, i.e., according to one marker in 8 cases (CD34-/+/Vim-2-/+ or CD34++/Vim-2++) and two markers in 4 cases (CD34-/+/Vim-2++). Since these data indicate that AML and normal progenitors were frequently distinguishable, we then determined the potential utility of these phenotypic dissimilarities for detection of minimal disease. Artificial mixtures of normal bone marrow and minimal numbers (0.1-1%) of AML cells were prepared. Based upon the phenotypic discrepancies, AML metaphases were successfully demonstrated in these mixtures following cell sorting and culture. Thus, it appears that minimal numbers of AML mitoses can be identified with an approximate 10(-2) to 10(-3) sensitivity by taking advantage of differential coexpression of surface antigens.

Genomic organization of the translocations (8;14) and (14;18) in a new lymphoma cell line.

We generated a new lymphoma cell line carrying the translocations (8;14) and (14;18) and studied the genomic organization and expression of the BCL-2 and MYC genes. Polymerase chain reaction (PCR) and Southern analysis showed that the breakpoints of t(14;18) were located in the major breakpoint region (mbr) of the BCL-2 gene and just 5' of JH6 in the IgH locus. The breakpoints of the t(8;14) were located upstream of exon 2 in the non-coding region of the MYC gene and near the switch region of the IgH locus. Both IgH loci were involved in chromosomal translocations resulting in the absence of a functional B-cell receptor. Normal BCL-2 and truncated MYC transcripts were detected in these cells. The BCL-2 protein was expressed.

Induction of granulocytic maturation in acute myeloid leukemia by G-CSF and retinoic acid.

AML cells were cultured free of serum with G-CSF in combination with all-trans-retinoic acid (RA), prostaglandin E2 or 8-bromocyclic AMP to see whether the maturation blockade of these cells could be overcome. The combination G-CSF + RA was most effective in inducing morphologic maturation, i.e. in 7/10 cases. Morphological alterations in response to G-CSF + RA indicated progression of the cells along the granulocytic pathway towards metamyelocytes and granulocytes. However, morphologically mature AML cells remained negative for myeloperoxidase and Sudan black stainings, indicators of granulocytic maturation. Chloracetate esterase positivity and CD15 membrane antigens became expressed on cultured AML cells, i.e. on unstimulated and G-CSF/RA exposed blasts. Ingestion of latex beads and reduction of nitroblue tetrazolium salt occurred in cultured AML cells regardless of the presence of inducers. In almost all cases clonogenic cells persisted after exposure to G-CSF + RA suggesting that subpopulations of immature cells escaped the action of these inducers. Thus although G-CSF + RA were capable of inducing maturation of AML cells along the granulocytic lineage, maturation was incomplete and the effect was evident in a subfraction of the cells only.

MHC- and Igh-unrestricted interaction between L3T4+, Lyt-2- Ts inducer cells and L3T4-, Lyt-2+ Ts effector cells is required for T cell-dependent suppression of DTH to histocompatibility antigens.

This paper describes the characteristics of T suppressor inducer (Ts ind) cells which can interact with T suppressor effector (Ts eff) cells and thereby can account for suppression of delayed-type hypersensitivity (DTH) to alloantigens. Adoptive transfer of spleen cells from mice intravenously (i.v.) injected with allogeneic spleen cells one day earlier induced an antigen-specific state of suppression in the recipients. This became apparent when DTH was induced by subcutaneous (s.c.) immunization of the recipients three days after transfer. The induction of suppression after adoptive transfer of spleen cells required Thy-1+, L3T4+, Lyt-1+2- cells. These cells that by themselves did not exert a suppressive effect induced a state of suppression in recipient mice by activation of recipient-type Ts eff cells. Therefore, the former cell type was classified as Ts ind cell. When athymic nude mice were used as recipients, Lyt-2+ precursors of Ts eff cells had to be transferred together with the Ts ind cells to induce a state of suppression in these mice. The Ts ind cells could activate Ts eff cells in MHC- and Igh-incompatible recipients. The results are discussed in relation to previously described immunoregulatory T cell pathways.

Lipopolysaccharide-induced suppression of graft-versus-host reactivity in mice.

Reconstitution of lethally irradiated mice with spleen cells from donors that had been treated with lipopolysaccharide (LPS) intravenously and allogeneic spleen cells subcutaneously leads to a suppressed anti-host delayed-type hypersensitivity (DTH). Either donor injection alone proved to be ineffective. The state of suppression appeared to be antigen-specific, but, depending on the experimental conditions, also anti-host DTH to third-party alloantigens could be suppressed. The suppression was mediated by a population of Thy-1- suppressor cells that could also be induced in athymic nude mice. The suppressor cells specifically adhered to anti-kappa-coated plastic plates, but were not adsorbed by passage through a Sephadex G-10 column. Thus, it appears that the combined donor treatment with LPS and allogeneic spleen cells induces a population of B cells that can suppress anti-host immune reactivity.

Suppression of antigraft immunity by preimmunization. V. Characterization of suppressor T memory cells.

We have previously shown that after intravenous (i.v.) immunization of mice with allogeneic spleen cells, two populations of suppressor T (Ts) cells may occur that can suppress delayed-type hypersensitivity (DTH) to alloantigens: Ts effector cells that transiently occur in the spleen, and long-lived, recirculating Ts cells that occur in thoracic duct lymph and can be transferred by parabiosis. In this study, we investigated whether the latter Ts cells fulfill the criteria for memory T lymphocytes, such as induction by doses of antigen lower than required for T effector cell induction, accelerated onset of activity after reactivation and an increased activity as compared with virgin T cells. The Ts cells accounting for the long-lasting state of suppression of DTH to alloantigens indeed fulfilled these criteria. These Ts memory cells displayed the Thy-1+, L3T4-, Lyt-1+2+ phenotype.

Lipopolysaccharide-induced suppression of DTH-reactivity to histocompatibility antigens. I. Kinetic aspects and specificity.

The effects of bacterial lipopolysaccharide (LPS) on the development of DTH-reactivity to alloantigens in mice were investigated. DTH to a particular set of alloantigens could be suppressed by treatment of responder mice with a single intravenous (i.v.) injection of 100 micrograms LPS and a simultaneous subcutaneous (s.c.) injection of the appropriate allogeneic spleen cells. The suppression lasted at least 60 days and affected the afferent limb of the DTH response as well as the efferent limb. The suppression could be adoptively transferred to naive syngeneic recipient mice by spleen cells, but not by immune serum, and proved to be antigen-specific. In spite of this specificity, the DTH-response against unrelated "third-party" alloantigens could be suppressed as well, provided the latter were presented during the induction-phase of DTH together with the alloantigens that had been used for the induction of suppression.

Genomic imprinting in testicular germ cell tumours.

Genomic imprinting refers to the parental origin-specific functional difference between the paternally and maternally-derived mammalian haploid genome. Normal embryogenesis depends on the presence of both a paternal and a maternal copy of particular chromosomal regions, containing the so-called imprinted genes. Genomic imprinting is established somewhere in the maturation from a primordial germ cell to a mature gamete, either spermatid or oocyte. We discuss the value of testicular cancers, especially those derived from the germ cell lineage, as a model to study erasement of the biparental pattern of genomic imprinting as present in the zygote and establishment of the paternal pattern during spermatogenesis. In addition, we will present data on the presence of X-inactivation in these cancers.

Cytogenetic evidence that carcinoma in situ is the precursor lesion for invasive testicular germ cell tumors.

A cytogenetic study of two cases of carcinoma in situ of the testis (CIS) and their adjacent invasive tumors, one a nonseminomatous germ cell tumor (NS) and one a seminoma (SE), revealed similarities in chromosomal pattern between the CIS and the invasive lesion in the same patient. These findings present for the first time cytogenetic evidence that CIS of the testis and its adjacent germ cell tumor are clonally related, which suggests that the CIS is indeed the precursor lesion of the invasive tumor.

Differential expression of SOX17 and SOX2 in germ cells and stem cells has biological and clinical implications.

Combined action of SOX and POU families of transcription factors plays major roles in embryonic development. In embryonic stem cells, the combination of SOX2 and POU5F1 (OCT3/4) is essential for maintaining the undifferentiated state by activating pluripotency-linked genes, and inhibition of genes involved in differentiation. Besides embryonic stem cells, POU5F1 is also present in early germ cells, primordial germ cells, and gonocytes, where it has a role in suppression of apoptosis. Here we demonstrate that SOX2 is absent in germ cells of human fetal gonads, and as expected carcinoma in situ (CIS), ie the precursor lesion of testicular germ cell tumours of adolescents and adults (TGCTs), and seminoma. Based on genome-wide expression profiling, SOX17 was found to be present, instead of SOX2, in early germ cells and their malignant counterparts, CIS and seminoma. Immunohistochemistry, western blot analysis, and quantitative RT-PCR showed that SOX17 is a suitable marker to distinguish seminoma from embryonal carcinoma, confirmed in representative cell lines. Aberrant SOX2 expression can be present in Sertoli cells when associated with CIS, which can be misdiagnosed as embryonal carcinoma. In conclusion, this study demonstrates the absence of SOX2 in human embryonic and malignant germ cells, which express SOX17 in conjunction with POU5F1. This finding has both diagnostic and developmental biological implications. It allows the identification of seminoma-like cells from embryonal carcinoma based on a positive marker and might be the explanation for the different function of POU5F1 in normal and malignant germ cells versus embryonic stem cells.