Much of the proteolysis that occurs during apoptosis is directed by caspases, a family of related cysteinyl proteases. A relatively small number of cellular proteins are targeted by caspases, yet their function is dramatically affected and apoptosis is triggered. Other proteases, such as granzymes and calpain, are also involved in the apoptotic signaling process, but in a much more cell type- and/or stimulus type-specific manner. At least three distinct caspase-signaling pathways exist; one activated through ligand-dependent death receptor oligomerization, the second through mitochondrial disruption, and the third through stress-mediated events involving the endoplasmic reticulum. These pathways also appear to interact to amplify weak apoptotic signals and shorten cellular execution time. Finally, defects in caspases contribute to autoimmune disease, cancer and certain neurological disorders.

PROCEDURE:To clarify whether the caspase 8 gene is involved in the pathogenesis of neuroblastoma (NB), we examined alterations of the caspase 8 gene in 15 NB, seven Ewing sarcoma (ES), and eight rhabdomyosarcoma (RMS) cell lines, using reverse transcription-polymerase chain reaction (RT-PCR) and RT-PCR single-strand conformation polymorphism (SSCP) analyses.RESULTS:The caspase 8 gene was not expressed in 11 (73%) of 15 NB cell lines, it was absent in only one of seven ES cell lines, but was present in all eight RMS cell lines examined. No mutations were detected in any cell lines examined.CONCLUSIONS:Inactivation of the caspase 8 gene is considered to be involved in the pathogenesis of NB, but not ES or RMS.

The human CASP8 gene, whose product is also known as caspase 8 and FLICE, encodes an interleukin-1β converting enzyme (ICE)-related cysteine protease that is activated by the engagement of several different death receptors. Caspase 8 is immediately recruited to the Fas receptor once it oligomerizes, and its protease activity is crucial for the apoptotic response generated by the resulting death-inducing signaling complex (DISC). We report here that the CASP8 gene contains at least 11 exons spanning ∼30kb on human chromosome band 2q33–34. This region of human chromosome 2 was previously reported as the location of the CASP10 gene, whose product is closely related to caspase 8. Chromosome 2 band q33–34 is also involved in tumorigenesis, with loss of heterogeneity (LOH) being reported in a number of tumors. We also report EcoRI and HindIII polymorphisms that may prove to be useful in disease analysis. Both caspases 8 and 10 contain long pro-domains with duplicated death effector domains (DEDs), as well as their corresponding cysteine protease catalytic domains. Thus, it appears that CASP8 and CASP10 have evolved by tandem gene duplication, much like the CASP1, CASP4 and CASP5 gene cluster on human chromosome 11q22.2–22.3.

The beta-globin locus control region is known to be a powerful erythroid-specific enhancer. In an attempt to produce immortalized erythroid percursor cells, transgenic mice have been generated with the simian virus (SV) 40 T antigen gene under the control of the locus control region. As previously reported, transgenic mice did not develop erythroleukaemia, but rather succumbed to insulinomas and poorly differentiated rhabdomyosarcomas. This paper describes additional mice containing this transgene that developed thymomas of the mixed epithelial/lymphocytic type, in which only the epithelial component expressed the T antigen. Epithelial cell lines have been established from these tumours. This system may be useful in future studies on the pathogenesis of thymomas and the function of thymic epithelial cells.

Transgenic mice harboring simian virus 40 large T antigen (Tag) gene fused to an erythroid-specific enhancer developed soft tissue sarcomas which expressed very high levels of T antigen. The Tag expression was not detectable in the animals' non-transformed tissues. While mice bearing several copies of the transgene developed tumors at an early age of 4-6 months, those with a single copy had a delayed onset of 10-16 months, and DNA analysis of their tumors showed amplification of the Tag transgene. Amplification of a Tag transgene has also been described previously in brain tumors. Our studies demonstrate that Tag transgene amplification is not restricted to a particular construct or a single tumor type. Therefore, this may be a general mechanism for Tag-mediated carcinogenesis, and our transgenic mouse system can be useful for elucidating the mechanisms that govern the amplification process of Tag sequences in vivo.

We have expressed the simian virus 40 (SV40) large T antigen oncogene in erythroid tissues of mice to test its ability to immortilize erythroid cells. A transgene construct was built in which the SV40 large T antigen structural gene was linked to erythroid-specific enhancer and promoter sequences. The enhancer employed was the human beta-globin family microlocus control region, and the promoter sequences were derived from the human beta-globin promoter. Transgenic mice were generated and they expressed T antigen in the bone marrow and spleen cells. Yet, no hematopoietic neoplasia arose in these mice. Instead, after a lag period of 2-6 months, the mice developed soft tissue sarcomas and pancreatic islet-cell tumors that expressed high levels of T antigen.

The beta-globin locus control region (LCR) confers a high level of erythroid-specific and copy-number-dependent expression to human globin genes in transgenic mice. Simian virus 40 T (tumor) antigen (Tag) with its own natural enhancer causes choroid plexus tumors in mice. We investigated the effect of the LCR on Tag gene expression, reasoning that mice harboring a LCR-Tag fusion gene might develop hematopoietic malignancies. To test this hypothesis we introduced an enhancerless Tag gene downstream of a LCR cassette into the germ lines of mice. The phenotypes of the transgenic mice depended on the copy number of the transgene. While mice with 1-2 copies matured normally, those with 3-7 copies developed rhabdomyosarcomas in different anatomic sites at high frequency and showed hyperplasia of the pancreatic islet cells which progressed to pancreatic islet tumors. In addition, the mice bearing 7 copies of the transgene had hypoglycemia and were stunted in growth. Mice with more than 10 copies were markedly stunted in growth and died within 2-4 weeks. Tag expression was detected at high levels in the mouse tumors but not in any other tissues, including the hematopoietic cells.

An immortalized xeroderma pigmentosum cell line belonging to the complementation group D (XP-D) was transfected with a normal human cDNA clone library constructed in a mammalian expression vector. Following UV-irradiation-selection, a transformant having a stable, partially UV-resistant phenotype was isolated. A transfected cDNA of partial length was rescued from the transformant's cellular DNA by in vitro amplification, using expression-vector specific oligonucleotides as primers in a polymerase chain reaction (PCR). Expression of this cDNA complemented the UV sensitivity of the XP-D cell line to the UV-resistance levels characteristic of the primary transformant. The nucleotide sequence of the cDNA was determined. The deduced protein identified the cDNA as encoding for the beta subunit of casein kinase II (CKII-β). Similar to the effect exerted by the truncated CKII-β cDNA, expression of a cDNA clone encompassing the complete translated region of CKII-β leads to XP-D cells partially resistant to UV-irradiation. However, transfection of CKII-β cDNA could also partially complement the UV-sensitivity of a xeroderma pigmentosum cell line belonging to group C (XP-C). Analysis by Southern, Northern and RNAase mismatch cleavage techniques did not reveal any functional defect in the CKII-β gene of cell lines derived from either 7 XP-D or 10 XP-C families. We therefore consider it unlikely that either the XP-D or the XP-C DNA repair deficiency is associated with a defect in the beta subunit of casein kinase II. Nevertheless, our findings suggest the possibility that the cell's response to DNA damage is modulated by CKII-dependent protein phosphorylation.

A xeroderma pigmentosum (XP) cell line from complementation group C has been complemented to attain ultraviolet (UV) resistance and DNA repair proficiency, by transfection with a human expression cDNA library, followed by selection to UV resistance. We now show that the transfected cDNAs can be rescued from cellular DNA of a secondary transformant by its in vitro amplification using expression-vector-specific oligodeoxyribonucleotides as primers in a polymerase chain reaction. The amplified cDNAs were cloned into a mammalian expression vector. Their transfection into XP cells identified a single cDNA which specifically complemented the UV sensitivity of a group-C-derived cell line to the same partial UV-resistance levels exhibited by the transformant from which the cDNAs were rescued.

In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion.