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.

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.

The PTEN gene in locally progressive prostate cancer is preferentially inactivated by bi-allelic gene deletion.

PTEN is frequently inactivated during the development of many cancers, including prostate cancer, and both bi-allelic and mono-allelic PTEN inactivation may contribute to tumorigenesis. PTEN mutations in clinical cancer specimens can easily be recorded but mono- or bi-allelic gene deletions are often difficult to assess. We performed a comprehensive study to detect PTEN inactivation in 40 locally progressive clinical prostate cancer specimens obtained by transurethral resection of the prostate, utilizing a variety of complementary technical approaches. The methods to detect PTEN deletion included allelotype analysis, dual-colour FISH and array-based CGH. We also applied a novel semi-quantitative approach, assessing the PTEN-WT (wild-type): PTEN-Psi (pseudogene) ratio (WPR). Structural analysis of PTEN was performed by single-strand conformational polymorphism (PCR-SSCP) and sequencing. PTEN protein expression was assessed by immunohistochemistry. Our data predict complete PTEN inactivation in 12 samples (30%), nine of these by bi-allelic deletion. Loss of one PTEN copy was also detected by several methodologies but the number could not be accurately assessed. Immunohistochemistry indicated the absence of PTEN protein in 15 samples, and heterogeneous expression of the protein in eight tumours. Taken together, these data show that bi-allelic deletion is a major mechanism of PTEN inactivation in locally progressive prostate cancer.

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.

Reactivity of germ cell maturation stage-specific markers in spermatocytic seminoma: diagnostic and etiological implications.

It is generally accepted that testicular seminomas and spermatocytic seminomas have separate pathogeneses, although the origin of these two types of germ cell tumors of the adult testis remains a matter of debate. Although an embryonic germ cell origin seems to be most likely for seminomas, a spermatogonia-spermatocyte origin has been suggested for spermatocytic seminoma. To shed more light on the etiology of spermatocytic seminomas, we undertook an immunohistochemical and molecular approach using SCP1 (synaptonemal complex protein 1), SSX (synovial sarcoma on X chromosome), and XPA (xeroderma pigmentosum type A) as targets. Although a stage-specific expression pattern has been reported for SCP1 and SSX in normal spermatogenesis, we demonstrate here that it also exists for XPA. In fact, immunohistochemistry shows that the proteins of SCP1 and XPA are specifically present in the stage of primary and pachytene spermatocytes. In contrast, SSX was found in spermatogonia and primary spermatocytes, as well as in germ cells, from at least the 17th week of intrauterine development onward. Although no protein encoded by any of these genes was detected in tumor cells of a series of testicular seminomas, all tested spermatocytic seminomas were positive, in agreement with expression analysis. These data support the model that seminomas originate from an embryonic germ cell, and they imply that the cell of origin of spermatocytic seminomas is at least capable of maturing to the stage of spermatogonia-pachytene spermatocyte.

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.

Restricted 12p amplification and RAS mutation in human germ cell tumors of the adult testis.

Human testicular germ-cell tumors of young adults (TGCTs), both seminomas and nonseminomas, are characterized by 12p overrepresentation, mostly as isochromosomes, of which the biological and clinical significance is still unclear. A limited number of TGCTs has been identified with an additional high-level amplification of a restricted region of 12p including the K-RAS proto-oncogene. Here we show that the incidence of these restricted 12p amplifications is approximately 8% in primary TGCTs. Within a single cell formation of i(12p) and restricted 12p amplification is mutually exclusive. The borders of the amplicons cluster in short regions, and the amplicon was never found in the adjacent carcinoma in situ cells. Seminomas with the restricted 12p amplification virtually lacked apoptosis and the tumor cells showed prolonged in vitro survival like seminoma cells with a mutated RAS gene. However, no differences in proliferation index between these different groups of seminomas were found. Although patients with a seminoma containing a homogeneous restricted 12p amplification presented at a significantly younger age than those lacking it, the presence of a restricted 12p amplification/RAS mutation did not predict the stage of the disease at clinical presentation and the treatment response of primary seminomas. In 55 primary and metastatic tumors from 44 different patients who failed cisplatinum-based chemotherapy, the restricted 12p amplification and RAS mutations had the same incidence as in the consecutive series of responding patients. These data support the model that gain of 12p in TGCTs is related to invasive growth. It allows tumor cells, in particular those showing characteristics of early germ cells (ie, the seminoma cells), to survive outside their specific microenvironment. Overexpression of certain genes on 12p probably inhibits apoptosis in these tumor cells. However, the copy numbers of the restricted amplification of 12p and K-RAS mutations do not predict response to therapy and survival of the patients.

Comparative genomic hybridization of microdissected samples from different stages in the development of a seminoma and a non-seminoma.

Human testicular germ cell tumours (TGCTs) of adolescents and adults, both seminomas and non-seminomas, originate from intratubular germ cell neoplasia (IGCN). Comparative genomic hybridization (CGH) was applied to microdissected samples from different stages of the development of a seminoma and a mixed non-seminoma, including IGCN of both. The different stages of the seminoma development, namely IGCN, intratubular and invasive seminoma, showed a very similar pattern of chromosomal imbalances, including gains of parts of 7, 8, 12,14, and X, and losses of parts of 3, 4, 5, 10, 11, 12q, 16, 18, 22, and Y. A more heterogeneous pattern was found for the non-seminoma. Some aberrations were present only in IGCN, or in IGCN and in all invasive components (gains of parts of 1q, 17, 19p, 20q, and 22, and losses of parts of 4, 5, 9p, 13, and 18q), while others were present in a less consistent pattern. These are the first reported CGH data from different stages in the development of TGCTs. Although only two cases were studied, the results suggest that particular numerical changes of (parts of) chromosomes are involved in the early development and progression of this cancer.

Lack of Bcl10 mutations in testicular germ cell tumours and derived cell lines.

Aberrations within Bcl10, a gene involved in execution of apoptosis, has most recently been found in a variety of cancers, including cell lines of testicular germ cell tumours of adolescents and adults (TGCTs). To study this in more detail, we screened exons 2 and 3 of this gene for mutations in a larger series of cell lines as well as primary TGCTs by single-strand conformation polymorphism and endonuclease restriction analysis. Because no aberrations were detected, we conclude that inactivation of Bcl10 by mutation is at least far less important in the development of TGCTs than proposed.

Detection of human endogenous retrovirus type K-specific transcripts in testicular parenchyma and testicular germ cell tumors of adolescents and adults: clinical and biological implications.

Testicular germ cell tumors (TGCTs) of adolescents and adults have been shown to contain proteins of the human endogenous retrovirus type K family. In a recent study, expression of these retroviral sequences was confirmed using in situ hybridization, which also showed expression in carcinoma in situ, the precursor of all TGCTs. Because of the clinical significance of a test for early diagnosis of TGCTs, we studied whether expression of human endogenous retrovirus type K genes could be an informative parameter. Therefore, we investigated TGCTs of various histologies and testicular parenchyma with and without carcinoma in situ using reverse transcription-polymerase chain reaction for expression of the gag, env, and prt genes. The gag and prt genes were expressed in all samples tested. The env transcripts were not found in TGCTs showing somatic differentiation only but could be detected in most normal testicular parenchyma samples. Therefore, detection of human endogenous retrovirus type K transcripts cannot be used for early diagnosis of TGCTs. Simultaneous expression of multiple gag sequences was found both in normal parenchyma and TGCTs, and we demonstrated that expression of gag sequences with an extra G, necessary to generate a functional protein, was not limited to TGCTs.

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.

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.

X inactivation in human testicular tumors. XIST expression and androgen receptor methylation status.

In female mammalian cells, inactivation of one of the X chromosomes compensates the increased dosage of X-linked genes as compared with their male counterparts. This process is initiated by the X-inactive specific transcripts of the xist/XIST gene in cis, resulting in methylation of specific sites of genes to be silenced. However, in male germ cells, X inactivation is established by xist/XIST expression only. We investigated the X inactivation pattern in human testicular tumors of different histogenesis by analysis of XIST expression and methylation of the androgen receptor gene. XIST was expressed only in tumors derived from the germ cell lineage with supernumerical X chromosomes: seminomas, nonseminomas, and spermatocytic seminomas. Although low expression was present in testicular parenchyma with spermatogenesis, XIST was expressed at a higher level in parenchyma with carcinoma in situ, the precursor lesion of seminomas and nonseminomas. Despite the consistent expression of XIST in germ-cell-derived tumors with gain of X chromosomes, methylation of the androgen receptor gene was present in all differentiated but only in a proportion of the undifferentiated nonseminomas. This differential pattern of methylation was also found in a number of representative cell lines. Our data indicate that the counting mechanism resulting in X inactivation is functional in testicular cancers of different histogenesis. Moreover, the differentiation-dependent pattern of X inactivation as reported during normal development in the case of multiple X chromosomes by methylation is retained in these tumors. We conclude therefore that X inactivation allows the excessive gain of X chromosomes found in germ-cell-derived tumors of the adult testis. In addition, this offers an interesting model to study the fundamental mechanisms of these processes.

Methylation similarities of two CpG sites within exon 5 of human H19 between normal tissues and testicular germ cell tumours of adolescents and adults, without correlation with allelic and total level of expression.

Testicular germ cell tumours (TGCTs) of adolescents and adults morphologically mimic different stages of embryogenesis. Established cell lines of these cancers are used as informative models to study early development. We found that, in contrast to normal development, TGCTs show a consistent biallelic expression of imprinted genes, including H19, irrespective of histology. Methylation of particular cytosine residues of H19 correlates with inhibition of expression, which has not been studied in TGCTs thus far. We investigated the methylation status of two CpG sites within the 3' region of H19 (exon 5: positions 3321 and 3324) both in normal tissues as well as in TGCTs. To obtain quantitative data of these specific sites, the ligation-mediated polymerase chain reaction technique, instead of Southern blot analysis, was applied. The results were compared with the allelic status and the total level of expression of this gene. Additionally, the undifferentiated cells and differentiated derivatives of the TGCT-derived cell line NT2-D1 were analysed. While peripheral blood showed no H19 expression and complete methylation, a heterogeneous but consistent pattern of methylation and level of expression was found in the other normal tissues, without a correlation between the two. The separate histological entities of TGCTs resembled the pattern of their nonmalignant tissues. While the CpG sites remained completely methylated in NT2-D1, H19 expression was induced upon differentiation. These data indicate that methylation of the CpG sites within exon 5 of H19 is tissue dependent, without regulating allelic status and/or total level of expression. Of special note is the finding that, also regarding methylation of these particular sites of H19, TGCTs mimic their non-malignant counterparts, in spite of their consistent biallelic expression.

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.

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.

Origin and biology of a testicular Wilms' tumor.

A pure triphasic testicular Wilms' tumor, without teratomatous elements, was studied using multiple techniques. Carcinoma in situ (CIS), the characteristic precursor of testicular germ cell tumors of adults (TGCTs), was found in the adjacent parenchyma. Flow cytometric analysis showed a single hypotriploid tumor stem line. Karyotyping of the tumor revealed some numerical and structural abnormalities, including an i(12p), the chromosomal marker of TGCTs. In situ hybridization supported the karyotypic findings, and showed a similar numerical distribution in CIS and the tumor. Molecular analysis of the tumor illustrated that all short arms of chromosome 12, including i(12p), were of maternal origin. No 12q deletions were detected. In spite of complete loss of the paternal 11p13 band, the zinc finger regions and exons 2 and 6 of the WT1 gene contained no aberrations. Therefore, this tumor suppressor gene is not inactivated due to aberrations in the studied regions. In addition, all four WT1 alternative transcripts were expressed in the tumor. No aberrations were found in chromosomal bands 11p15.5, 16q22.1, and 16q24. Both parental alleles of the human imprinted genes H19 and IGF2 were expressed in the tumor. This is the first report on the chromosomal and molecular characterization of an extrarenal Wilms' tumor. Its germ cell origin was unequivocally demonstrated.

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.

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.

Introduction of the human interleukin-6 (IL-6) receptor in murine IL-3-dependent hematopoietic cells restores responsiveness to IL-6.

To create experimental systems that facilitate studies aimed at the responsiveness of hematopoietic progenitors to interleukin (IL)-6 in combination with IL-3, we introduced the human IL-6 receptor (hIL-6R) into the IL-3-dependent cell line 32D. For this purpose, a retroviral vector containing the hIL-6R cDNA was constructed. 32D parental cells did not respond to IL-6, neither alone nor in combination with increasing concentrations of IL-3, and did not express detectable numbers of IL-6R as determined by 125I-IL-6 binding. 32D/hIL-6R cells expressed high-affinity IL-6 binding and responded synergistically to IL-6 in combination with suboptimal amounts of IL-3 in DNA synthesis assays. In addition, IL-6 promoted the short-term survival of IL-3-responsive clonogenic 32D/hIL-6R cells. On the other hand, although introduction of hIL-6R resulted in the formation of high-affinity IL-6 receptor structures in the IL-2-dependent thymocyte cell line CTLL, CTLL/hIL-6R cells did not respond to IL-6 in synergy with IL-2. We conclude that 32D cells possess the intracellular machinery permissive for IL-6 signal transduction. Murine IL-3-dependent cell lines with ectopic IL-6 receptors can serve as models for dissecting the molecular basis of IL-6 responses in primitive hematopoietic cells.