Isolation of single-copy human genes from a library of yeast artificial chromosome clones.

A recently developed cloning system based on the propagation of large DNA molecules as linear, artificial chromosomes in the yeast Saccharomyces cerevisiae provides a potential method of cloning the entire human genome in segments of several hundred kilobase pairs. Most application of this system will require the ability to recover specific sequences from libraries of yeast artificial chromosome clones and to propagate these sequences in yeast without alterations. Two single-copy genes have now been cloned from a library of yeast artificial chromosome clones that was prepared from total human DNA. Multiple, independent isolates were obtained of the genes encoding factor IX and plasminogen activator inhibitor type 2. The clones, which ranged in size from 60 to 650 kilobases, were stable on prolonged propagation in yeast and appear to contain faithful replicas of human DNA.

Genetic transfer and expression of reconstructed yeast artificial chromosomes containing normal and translocated BCL2 proto-oncogenes.

The goal of this study was to determine whether it will be feasible to study the expression of a large, human gene, such as the BCL2 proto-oncogene, by DNA transfection. The BCL2 proto-oncogene is 230 kb in size and is deregulated in tumor cells by translocation into the immunoglobulin heavy-chain locus. Yeast artificial chromosomes (YACs) containing the human BCL2 gene were altered by homologous recombination in Saccharomyces cerevisiae to yield replicas of the normal and translocated alleles. Constructions containing either allele and ranging in size from 360 to 800 kb were integrated stably into a mouse tumor line. Fifty-eight percent of the clones contained a copy of the entire YAC insert. Over 50% of these clones expressed appropriate levels of human BCL2 RNA and protein. These studies suggested that the expression of large human genes and their pathologic rearrangements can be studied by transfection techniques employing YACs propagated in S. cerevisiae.

Suppression of a squamous cell carcinoma (SCC)-related serpin, SCC antigen, inhibits tumor growth with increased intratumor infiltration of natural killer cells.

Squamous cell carcinoma (SCC) antigen (SCCA), a member of the ovalbumin serine proteinase inhibitor family, serves as a circulating marker of squamous cell carcinoma (SC). One of the SCCAs, SCCA1, has been suggested to play a role in the attenuation of apoptosis in vitro and in the augmentation of tumor growth in vivo. In the present study, the infection of a SCC cell line (SKG IIIa) with recombinant retrovirus that expressed the antisense SCCA mRNA suppressed expression of SCCA in vitro. Local administration of this retrovirus into tumors by inoculation in nude mice suppressed tumor growth. Treatment of tumor tissue in vivo is also associated with increased numbers of apoptotic tumor cells and large mononuclear cells in the tumor. To test the possible role of SCCA in the infiltration of large mononuclear cells, we analyzed the effect of SCCA1 on migration of natural killer (NK) cells induced by monocyte-chemoattractant protein-1 in vitro. SCCA1 suppressed migration of NK cells completely, and this inhibitory effect was lost by mutation of the reactive site loop of SCCA1. These results suggest that antisense SCCA may suppress the growth of SCC in vivo not only by the augmentation of intracellular apoptosis but also by the increased infiltration of NK cells into the tumor.

Genomic cloning, mapping, structure and promoter analysis of HEADPIN, a serpin which is down-regulated in head and neck cancer cells.

Headpin is a novel serine proteinase inhibitor (serpin) that is down-regulated in squamous cell carcinoma of the oral cavity and in squamous cell carcinoma cell lines of the head and neck. Using a panel of 18q21.3 YAC clones, we mapped and cloned the HEADPIN gene. The gene spans 10 kb and is composed of eight exons and seven introns. The genomic structure is identical with some other ovalbumin serpins (ov-serpins) in terms of the numbers, position and phasing of the intron/exon boundaries. HEADPIN was mapped within the serpin cluster in 18q21.3 between MASPIN and SCCA2 as follows: cen-MASPIN-HEADPIN-SCCA2-SCCA1-tel. The transcription start site was determined and the promoter activity of the 5'-flanking region was analyzed. Luciferase promoter assays in HaCaT cells showed that the -432 to -144 nucleotide region has functional promoter activity. The activity of the promoter/enhancer was not observed in head and neck cancer cell lines TU167 and UMSCC1 which lack headpin expression. These data suggest that the differential expression of headpin in normal and carcinoma-derived cells is regulated at the transcriptional level. Understanding the genomic organization and transcriptional regulation of the ov-serpins clustered within 18q21. 3 provides a critical framework for assessing their potential role in cancer.

Rapid detection of lymphoma-specific translocations in interphase nuclei of non-Hodgkin's lymphoma by fluorescence in situ hybridization.

We have recently developed a method to detect tumor-specific rearrangement of the IgH gene in interphase nuclei by fluorescence in situ hybridization. Tumor-specific IgH gene rearrangement is equivalent to 14q32.33 translocation. Using this approach, we detected 14q32.33 translocation in 29 of 70 patients with B-cell non-Hodgkin's lymphoma (NHL). Chromosome t(3;14) was found in 10 of these 29 patients, and were demonstrated as a fusion signal of BCL6 and VH gene probes in interphase nuclei. Furthermore, in another series of 11 patients and a NHL cell line, we demonstrated t(14;18) and t(11;14) in interphase and metaphase cells with a combination of BCL2 (or PRAD1) with IgH gene probes. Interphase FISH with lymphoma-associated gene probes is a rapid procedure for cytogenetic diagnosis of B-cell NHL.

Structure and sequence of human M/NEI (monocyte/neutrophil elastase inhibitor), an Ov-serpin family gene.

Human monocyte/neutrophil Elastase Inhibitor (M/NEI) is a proteinase inhibitor that regulates the activity of the neutrophil proteases: elastase, cathepsin G and proteinase-3. Evidence indicates that M/NEI belongs to the Ov-serpin family (ovalbumin-related serpins), functionally diverse proteins with shared structural features. Recombinant lambda phage clones were isolated that encompass the full-length M/NEI gene plus upstream and downstream regions. The gene, 9.5kb long, consists of 7 exons and 6 introns. The 5' transcription start site identified by primer extension corresponds to a 60bp exon 1; the translation start site is in exon 2. Southern blots established a gene copy number of one. The 3' untranslated region (UTR) contains three AATAAA/AATTAA sites; these were shown to function as alternative polyadenylation signals. A 14-nucleotide upstream motif including the atypical TATA box TATAAGAG otherwise occurs only twice in GenBank, in the genes encoding neutrophil elastase and proteinase-3, target proteases inhibited by M/NEI. Comparison of M/NEI and previously characterized related genes strongly suggests that all Ov-serpins, despite a difference in chromosomal localization and exon number, nonetheless, share a common basic gene structure.

Co-expression of the squamous cell carcinoma antigens 1 and 2 in normal adult human tissues and squamous cell carcinomas.

Squamous cell carcinoma antigen (SCCA) serves as a serological marker for advanced squamous cell carcinomas (SCCs) and as an indicator of therapeutic response. Recent molecular studies show that the SCCA is transcribed by two almost identical tandemly arrayed genes, SCCA1 and SCCA2. These genes are members of the high molecular weight serine proteinase inhibitor (serpin) superfamily. Although SCCA1 and SCCA2 are 92% identical at the amino acid level, they have distinct biochemical properties. Paradoxically, SCCA1 is an inhibitor of papain-like cysteine proteinases, such as cathepsins L, S, and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, cathepsin G, and mast cell chymase. Using a new set of discriminatory monoclonal antibodies (MAbs) and polymerase chain reaction (PCR) assay, we showed that SCCA1 and SCCA2 were co-expressed in the suprabasal layers of the stratified squamous epithelium of the tongue, tonsil, esophagus, uterine cervix and vagina, Hassall's corpuscles of the thymus, and some areas of the skin. SCCA1 and SCCA2 also were detected in the pseudo-stratified columnar epithelium of the conducting airways. Examination of squamous cell carcinomas of the lung and head and neck showed that SCCA1 and SCCA2 were co-expressed in moderately and well-differentiated tumors. Moreover, there was no differential expression between these SCCA "isoforms" in normal or malignant tissues. In contrast to previous studies, these data indicated that the expression of SCCA1 and SCCA2 was not restricted to the squamous epithelium and that these serpins may coordinately regulate cysteine and serine proteinase activity in both normal and transformed tissues.

Systemic antibody responses of different species following ingestion of soluble protein antigens.

Following ingestion of bovine serum albumin (BSA), human infants and rabbits synthesize circulating anti-BSA. Using a similar protocol, the serum antibody response was examined in C3H mice offered 0.1, 1.0 or 6.0% BSA for 10 or 56 days. Sera from these animals contained similar amounts of antigen, but anti-BSA was undetectable. Half the animals in each group were subsequently injected i.v. with 50.0 mg BSA. Mice fed 1.0 or 6.0% BSA were unresponsive to this challenge, but animals fed 0.1% BSA responded comparably to i.v. immunized, non-fed controls. The remaining animals in each group were injected s.c. with 1.0 mg BSA in adjuvant. Most of these mice produced circulating anti-BSA. However, these responses were significantly reduced relative to s.c. immunized, non-fed controls. Hyporesponsiveness again was least after 0.1% BSA ingestion. CF1 mice and BN, HCR and HCS rats fed 0.1% BSA for 56 days also did not produce circulating antibodies during ingestion, and almost all were hyporesponsive after parenteral challenge. Suppression of specific antibody responses also was observed in C3H mice fed and challenged with diphtheria toxoid. The data suggest that under identical experimental conditions, ingestion of soluble protein antigens induces a state of systemic hyporesponsiveness in mice and rats in contrast to the systemic immunity demonstrated in human neonates and adult rabbits.

SCCA1 expression in T-lymphocytes peripheral to cancer cells is associated with the elevation of serum SCC antigen in squamous cell carcinoma of the tongue.

Squamous cell carcinoma (SCC) antigen has been used for the management of SCC arising in various cites including head and neck region. However, the true mechanism of the elevation of this protein in the serum of patients with SCC is still unknown. SCC antigen belongs to the superfamily of serine protease inhibitors. Recently, molecular studies show that serum SCC antigen is transcribed by two nearly identical genes (SCCA1 and SCCA2), and is mainly produced by SCCA1. The objective of this study is to clarify the mechanism of the elevation of SCC antigen in oral tongue SCC patients and to identify cells histologically, which are responsible for serum SCC antigen production. In this study, we examined SCCA1 expression in a series of four head and neck SCC (HNSCC) cell lines, and found that all expressed equal to low SCCA1 protein as compared with the normal human oral keratinocyte. Using the double immunohistochemical technique to examine the expression pattern of SCCA1 in 86 cases of oral tongue squamous cell carcinoma, SCCA1 immunostaining was observed in the cytoplasm of cancer cells and T-lymphocytes peripheral to cancer cells. We also compared the clinicopathological features including serum SCC antigen level of the oral tongue SCC cases with the immunohistochemical SCCA1 expression pattern, and found that elevated serum SCC antigen level was significantly correlated with SCCA1 expression not in cancer cells, but in T-lymphocytes peripheral to cancer cells. These results suggest that T-lymphocytes peripheral to cancer cells may be responsible for serum SCC antigen production in HNSCC patients.

Gene expression in activated brain microglia: identification of a proteinase inhibitor that increases microglial cell number.

Microglia, the intrinsic immune cells of the central nervous system, are activated in a variety of inflammatory brain diseases in which they play a pathogenetic role. However, mechanisms underlying activation are largely unknown. To begin elucidating molecular mechanisms associated with activation, we characterized the pattern of gene expression in virtually pure dissociated microglial cultures, using RT-PCR differential display. Microglia were activated with bacterial lipopolysaccharide (LPS), a traditional stimulant, and the profile of gene expression was compared to that in basal, control cultures. Activation resulted in altered expression of six genes. The cDNAs were isolated, sequenced and characterized. Homology searches identified three novel genes, and two that exhibited very high sequence similarity to the gene encoding squamous cell carcinoma antigen (SCCA). SCCA (1 and 2) are tandemly arranged genes that encode two serine proteinase inhibitors (serpins). SCCA has been detected exclusively in cancer cells, and is a plasma marker for squamous cell carcinoma. Immunoblot analysis indicated that gene expression was accompanied by a 5-fold increase in the synthesis of SCCA protein in LPS-activated microglia. To assess potential biological actions of the SCCA serpins, SCCA1 protein was added to cultures. SCCA1 altered microglial morphology, and elicited a dramatic, 5-fold increase in cell number within 72 h. The effects appeared to be cell-specific, since the protein had no effect on other cell types: cortical astrocytes and neurons from cortex or basal forebrain were unaffected. We tentatively conclude that SCCA1 may play a cell-specific role in increasing cell number, a critical early step in microglial activation and brain inflammation. More generally, differential display of genes in the microglial model system may help define patterns of expression associated with CNS disease, thereby identifying pathogenetic mechanisms and new therapeutic targets.

Development of specific monoclonal antibodies and a sensitive discriminatory immunoassay for the circulating tumor markers SCCA1 and SCCA2.

The squamous cell carcinoma antigen (SCCA) serves as a serologic marker for advanced squamous cell carcinomas (SCC) of the uterine cervix, lung, esophagus, head and neck and vulva. Elevations in serum levels of SCCA following treatment for SCC correlate with tumor relapse or metastasis. Recent molecular studies show that SCCA is transcribed by two nearly identical genes (SCCA1 and SCCA2) that encode for members of the high molecular weight serine proteinase inhibitor (serpin) family. Despite a high degree of similarity in their amino acid sequences, SCCA1 and SCCA2 have distinct biochemical properties: SCCA1 is an inhibitor of papain like cysteine proteinases, such as cathepsins (cat) L, S and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, catG and mast cell chymase. In this paper, we report the generation and characterization of anti-SCCA1 and anti-SCCA2 specific monoclonal antibodies (MAbs). Using these MAbs, we developed an enzyme-linked immunoassay (ELISA) that discriminated between SCCA1 and SCCA2 without any cross-reaction. This assay measured both the native and complexed forms of SCCA1 and SCCA2. The sensitivity of detection of SCCA1 and SCCA2 assays were 0.17 ngml(-1) and 0.19 ngml(-1), respectively. Mean inter- and intra-assay coefficients of variation were 12.1% and 9.9% for SCCA1 assay and 12% and 8.8% for SCCA2 assay, respectively. Recovery and parallellism studies indicated that SCCA1 and SCCA2 were detected in the plasma and amniotic fluids without any major interference by the biologic fluid components. This assay provides a simple and accurate procedure for the quantitation of total SCCA1 and SCCA2.

Intracellular and extracellular serpins modulate lung disease.

An imbalance between peptidases and their inhibitors leads to pulmonary disease. Imbalances occur in the adult and the neonate at risk for a specific set of lung pathologies. Serpins (serine peptidase inhibitors) make up the major source of antipeptidase activity in the lung. The purpose of this review is to describe the serpin mechanism of inhibition, their roles in the normal and pathological lung and their potential as therapeutic agents.

Building larger YACs by recombination.

Despite the relatively large cloning capacity of YACs, many genomic regions or individual genes are not cloned intact, but are represented as a collection of overlapping clones or contigs. Fortunately, the relatively high frequency and fidelity of homologous recombination in Saccharomyces cerevisiae can be used to reconstruct intact genes within a single clone by splicing together overlapping DNA segments. This unit describes two protocols for carrying out such homologous recombination; one relies on the meiotic phase of the yeast cycle, while the other utilizes the mitotic phase of the yeast life cycle. Despite the relatively large cloning capacity of YACs, many genomic regions or individual genes are not cloned intact.

Specifically induced suppression of T cell and NK-like cytolytic activity by ingested soluble antigens.

The effect of feeding xenoserum (xs) on cytolytic cell activity induced by parenteral injection was examined in C3H/N mice. Spleen cells were cultured with xs and then assayed for cytolytic activity against a panel of 51Cr-labeled YAC-1, AKR-A, or P815 target cells. Prior feeding resulted in significant suppression of responses stimulated by injection and culture. The induction of these responses was antigen specific for xs whereas the effector stage represented polyclonal activation of cytolytic cells. Some effector cells were lysed by either anti-Lyt 2 or anti-NK- 1.2 and complement and some were blocked by anti-Lyt 2 or anti-T200 in the cytotoxicity assay. Thus, both cytolytic T and NK-like cells were suppressed by antigen feeding. Activity of TH cell-derived factors which enhance cytolytic activity ("promoter" factor, interferon, and interleukin 2) also was diminished in culture supernatants of cells from mice fed soluble antigens. The conclusion that polyclonal cytolytic responses induced by soluble antigen can be regulated by prior enteric stimulation is made.

STS map of genes and anonymous DNA fragments on human chromosome 18 using a panel of somatic cell hybrids.

Somatic cell hybrids containing different deleted regions of chromosome 18 derived from patients with balanced translocations or terminal deletions were used to create a deletion mapping panel. Twenty-four sequence-tagged sites (STSs) for 17 genes and 7 anonymous polymorphic DNA fragments were identified. These STSs were used to map the 24 loci to 18 defined regions of chromosome 18. Both ERV1, previously mapped to 18q22-q23, and YES1, previously mapped to 18q21.3, were found to map to 18q11.21-pter. Several genes previously mapped to 18q21 were found to be in the order cen-SSAV1-DCC-FECH-GRP-BCL2-PLANH2-tel. The precise mapping of genes to chromosome 18 should help in determining whether these genes may be involved in the etiology of specific chromosomal syndromes associated with chromosome 18. The mapping of the polymorphic loci will assist in the integration of the physical map with the recombination map of chromosome 18.

Chromosomal reassignment: YACs containing both YES1 and thymidylate synthase map to the short arm of chromosome 18.

The YES1 proto-oncogene was mapped previously to human chromosome band 18q21.3 by using isotopic in situ hybridization. Using yeast artificial chromosomes (YACs) as probes and fluorescence in situ hybridization, a strong signal was detected in the region corresponding to 18p11.3. Restriction digests confirmed that the YACs contained the YES1 gene and not other cross-hybridizing, protein-tyrosine kinases. In addition, these YACs were found to contain another 18p11.32 gene, thymidylate synthase. These genes were less than 50 kb apart. Collectively, these data suggest that YES1 maps to 18p11.32 rather than to 18q21.3.