Primary structure of the human 4F2 antigen heavy chain predicts a transmembrane protein with a cytoplasmic NH2 terminus

The human cell surface antigen 4F2 is a disulfide-linked heterodimer consisting of a glycosylated heavy chain and a nonglycosylated light chain. The antigen is ubiquitously expressed on proliferating cells but only in resting cells from certain tissues. Its function has been proposed to relate to cellular Ca2+/Na+ exchange. We describe the molecular cloning of the 4F2 heavy chain gene and cDNA by a gene transfer approach. Part of the gene was isolated from a genomic lambda library constructed with DNA of a secondary transfectant L cell line that expresses 4F2 antigen. A gene-specific probe derived from the phage inserts was used to isolate two full length cDNA clones. Both cDNA clones directed the expression of 4F2 antigen in transfected mouse L cells. The 4F2 antigen heavy chain gene specifies a 2.1-kilobase mRNA with an open reading frame coding for a 529-residue protein of 58 kDa. The protein lacks an NH2-terminal signal peptide but contains an internal transmembrane-spanning region and four potential glycosylation sites in its COOH-terminal domain. We predict that the 4F2 antigen heavy chain is a transmembrane protein with a cytoplasmic NH2 terminus of 81 amino acids. The antigen shows no homology to known protein sequences.     

Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid.

Biosynthesis of HLA class I antigens has been studied in a variant B-LCLxT-LCL hybrid, 174XCEM.T2. This cell line encodes HLA-A2 and -B5, but expresses only small amounts of A2 antigen and undetectable B5 antigen at the cell surface due to a mutation inactivating a trans-acting regulatory gene encoded within the class II region of the human major histocompatibility complex. Northern blot analysis with HLA-A- and HLA-B-specific probes shows that 174XCEM.T2 synthesizes quantities of A and B locus mRNA comparable with its class I antigen-positive parent cell line. Immune precipitation studies indicate that 174XCEM.T2 synthesizes normal HLA heavy chains and beta 2-microglobulin which fail to form dimers. The heavy chains are N-glycosylated normally, but processing of the glycan to the complex form does not occur. In addition, free heavy chains in this cell line are not phosphorylated. Thus, the majority of class I heavy chains in 174XCEM.T2 do not combine with beta 2-microglobulin, and are not processed or transported to the cell surface. As both subunits are synthesized in normal amounts, we propose that an additional molecule absent from 174XCEM.T2 and encoded by an HLA-linked gene is necessary for efficient assembly of class I antigen subunits.     

Enhanced production of hepatitis B surface antigen in NIH 3T3 mouse fibroblasts by using extrachromosomally replicating bovine papillomavirus vector.

We have constructed a recombinant pBR322 plasmid composed of a subgenomic transforming fragment of bovine papillomavirus DNA and the hepatitis B surface antigen gene from cloned hepatitis B virus DNA and used it for transfection of NIH 3T3 mouse fibroblasts. The transformed cells retain the plasmids in extrachromosomal form with a copy number of about 50 to 100 per cell. Expression of the hepatitis B surface antigen gene linked to bovine papillomavirus DNA is independent of its orientation relative to the bovine papillomavirus vector. Cell lines continuously secreting high amounts of hepatitis B surface antigen into the medium could be established. The antigen is released into the culture medium as 22-nm particles, having the same physical properties and constituent polypeptides as those found in the serum of hepatitis B virus-infected patients.     

The absence of β 2-microglobulin in Daudi cells: Active gene but inactive messenger RNA

The Daudi cell line is characterized by an absence of HLA antigen on its surface. This has been attributed to a lack of beta 2-microglobulin (beta 2m) while the heavy chain of HLA is present intracellularly. Karyotype analysis of Daudi cells has shown a deletion involving one of the beta 2-microglobulin alleles. It was generally believed that the absence of beta 2-microglobulin in that cell line resulted from an absence of expression of the remaining gene. We report here the unexpected finding of a normal amount of beta 2-microglobulin messenger RNA in Daudi cells. This was demonstrated by "Northern blot" hybridization with cDNA plasmid clones as a probe. This mRNA, however, when purified by hybridization-selection with beta 2-microglobulin plasmid DNA, is unable to function as messenger in protein synthesis and is therefore an inactive mRNA. The finding of a translationally inactive beta 2-microglobulin mRNA provides a new explanation for the absence of beta 2-microglobulin and therefore of HLA antigens in Daudi cells.     

Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.     

Distinct effects of interferon-gamma and MHC class I surface antigen levels on resistance of the K562 tumor cell line to natural killer-mediated lysis

Various investigators have examined the relationship between tumor cell susceptibility to natural killer (NK) cell lysis and the expression of HLA class I antigens on the tumor cell. There is controversy as to whether or not an inverse relationship exists, and if so, the basis of the relationship between these two phenomena remains undefined. To address these questions, the genomic clones for two HLA antigens were transfected into the erythroleukemia cell line K562, a cell line that is used as the standard to assess human NK and major histocompatibility complex (MHC) nonrestricted cytolysis. Susceptibility to NK lysis was not affected by the de novo expression of HLA antigens on the K562 after DNA mediated gene transfer. Interferon-gamma (IFN-gamma) treatment of K562 induced levels of MHC class I antigen surface expression comparable to those found on the transfected cells; however, the IFN-gamma-treated cells were resistant to NK lysis. When very high levels of surface HLA antigens were induced on the transfectants, a potential effect of class I MHC expression on K562 lysis could be discerned that was distinct from the resistance to NK lysis induced by IFN-gamma-treatment.     

Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines.

Using fluorescence in situ hybridization and Southern blot analysis, we show that three clonally derived cell lines transformed with bovine papillomavirus (BPV), including ID13, the cell line commonly employed for BPV replication studies, are heterogeneous populations having extensive cell-to-cell variation in both the distribution and amount of BPV DNA. Different subclones of ID13 were found to differ in the form and amount of BPV DNA they contain. Most subclones showed no detectable BPV sequences; some contained either extrachromosomal BPV molecules distributed throughout the nucleus or BPV sequences integrated at discrete chromosomal sites, while others contained both integrated and plasmid forms. The results of density gradient analysis of BPV DNA from individual homogeneous subclones showed replication of the extrachromosomal BPV plasmids in a random-choice mode. In all cell lines studied, the presence after one round of chromosomal DNA replication of unreplicated BPV DNA and of BPV DNA having two postreplicative strands was independent of the presence of high-BPV-copy-number ("jackpot") cells. Our results substantiate the earlier conclusion that extrachromosomal BPV molecules replicate randomly and not according to a once-per-cell-cycle mechanism.     

Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene.

Human papillomavirus (HPV) E6 and E7 oncogenes are expressed in the great majority of human cervical carcinomas, whereas the viral E2 regulatory gene is usually disrupted in these cancers. To investigate the roles of the papillomavirus E2 genes in the development and maintenance of cervical carcinoma, the bovine papillomavirus (BPV) E2 gene was acutely introduced into cervical carcinoma cell lines by infection with high-titer stocks of simian virus 40-based recombinant viruses. Expression of the BPV E2 protein in HeLa, C-4I, and MS751 cells results in specific inhibition of the expression of the resident HPV type 18 (HPV18) E6 and E7 genes and in inhibition of cell growth. HeLa cells, in which HPV gene expression is nearly completely abolished, undergo a dramatic and rapid inhibition of proliferation, which appears to be largely a consequence of a block in progression from the G1 to the S phase of the cell cycle. Loss of HPV18 gene expression in HeLa cells is also accompanied by a marked increase in the level of the cellular p53 tumor suppressor protein, apparently as a consequence of abrogation of HPV18 E6-mediated destabilization of p53. The proliferation of HT-3 cells, a human cervical carcinoma cell line devoid of detectable HPV DNA, is also inhibited by E2 expression, whereas two other epithelial cell lines that do not contain HPV DNA are not inhibited. Thus, a number of cervical carcinoma cell lines are remarkably sensitive to growth inhibition by the E2 protein. Although BPV E2-mediated inhibition of HPV18 E6 and E7 expression may contribute to growth inhibition in some of the cervical carcinoma cell lines, the BPV E2 protein also appears to exert a growth-inhibitory effect that is independent of its effects on HPV gene expression.     

Transfer of cloned human class I major histocompatibility complex genes into HLA mutant human lymphoblastoid cells.

Three new kinds of recombinant DNA constructs were used to transfer cloned human class I HLA genes (A2 and B8) into unique HLA mutant lymphoblastoid cells: pHeBo(x): a class I gene, "x," in plasmid vector pHeBo, which contains a hygromycin resistance gene and Epstein-Barr virus oriP element that sustains extrachromosomal replication; pHPT(x): gene x in a vector with a hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene; pHPTe(x): gene x in a vector with the HPRT gene and oriP element. Cell surface class I antigen expression was strong in transferents made with class I-deficient lymphoblastoid cell line mutants .144 (A-null), .53 (B-null), and .184 (A-null, B-null). Transferents expressing HLA-A2 were recognized specifically by HLA-A2-specific cytotoxic T lymphocytes. When introduced on either of the vectors with the Epstein-Barr virus oriP element, the class I gene replicated extrachromosomally and was lost at rates of 0.2 to 0.3 per cell division. When introduced with vector pHPT (lacking Epstein-Barr virus oriP), the B8 gene was inserted at different chromosomal locations. Introduction of the HLA-B8 gene failed to restore antigen expression by HLA-B-null mutant .174, providing evidence that, unlike mutants exemplified by .53, .144, and .184, some HLA antigen loss mutants are deficient in a trans-acting function needed for class I antigen expression. Of more general interest, the results obtained with HLA class I genes in vectors that replicate extrachromosomally suggest ways of relating genic expression to chromatin structure and function and of attempting to clone functional human centromeres.     

Association with beta 2-microglobulin controls the expression of transfected human class I genes

The genes encoding HLA-B27K and HLA-B27W were transfected into murine recipient cells. A monoclonal antibody HC-10, directed against free B-locus heavy chain, was the only reagent capable of efficiently detecting the HLA-B27 heavy chains in detergent lysates. These heavy chains were devoid of sialic acid. Trace amounts of HLA-B27 could be isolated with the anti-HLA-A,-B antibody W6/32, which reacts with the heavy chain beta 2-microglobulin complex. In marked contrast, HLA-A2 and -B7 genes, when transfected, yielded easily detectable amounts of antigen precipitable with W6/32, which carried the usual complement of sialic acids. Because the alpha 3 domains of HLA-B27 and HLA-B7 and the more COOH-terminal portions are identical in amino acid sequence, structural elements in the polymorphic alpha 1 and alpha 2 domains must control association of heavy chain with beta 2-microglobulin. Introduction of a human beta 2-microglobulin gene into L cells transfected with the HLA-B27 gene rescued the expression of HLA-B27 at the cell surface, as evidenced by reactivity with W6/32, surface staining, and the presence of sialic acid on the heavy chain.     

Tumorigenic transformation of murine keratinocytes by the E5 genes of bovine papillomavirus type 1 and human papillomavirus type 16.

To examine the biological properties of the bovine papillomavirus type 1 (BPV) and human papillomavirus type 16 (HPV16) E5 genes, each was cloned separately into a retroviral expression vector and helper-free recombinant viruses were generated in packaging cell lines. The BPV E5 retroviruses efficiently caused morphologic and tumorigenic transformation of cultured lines of murine fibroblasts, whereas the HPV16 E5 viruses were inactive in these assays. In contrast, infection of the p117 established line of murine epidermal keratinocytes with either the BPV or the HPV16 E5 retrovirus resulted in the generation of tumorigenic cells. Pam212 murine keratinocytes were also transformed to tumorigenicity by the HPV16 E5 gene but not by the gene carrying a frameshift mutation. These results establish that the HPV16 E5 gene is a transforming gene in cells related to its normal host epithelial cells.     

Stable expression of human tissue-type plasminogen activator regulated by beta-actin promoter in three human cell lines: HeLa, WI-38 VA13 and KMS-5

A high-level and stable expression system of human tissue-type plasminogen activator (t-PA) was accomplished in human cells by selecting a promoter and a host cell line. First, we have constructed two types of t-PA expression plasmids containing 3 kb of the human beta-actin promoter region or 0.3 kb of SV40 early promoter region and these plasmids were transfected into HeLa cells, respectively, and the resulting transfectants were found to secrete various amounts of t-PA derived from the plasmids to the culture media. Southern blot analysis revealed that the beta-actin promoter was more efficient than the SV40 early promoter with regard to the expression level per single copy of the t-PA gene in the transfected HeLa cells. Next, the t-PA expression plasmid containing the beta-actin promoter was also transfected into WI-38 VA13 cells, a human fibroblastic cell line, and KMS-5 cells, a human lymphoid cell line, in order to compare the expression ability of the promoter among these three cell lines. Some of the transfectants from both cell lines were also found to produce t-PA. It was also found that the expression levels in HeLa and WI-38 VA13 seemed to be more efficient than that in KMS-5.     

Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells.

Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.