Monoclonal antibody specific to bovine lens epithelial cell membrane: preparation of a lens epithelial cell-selective immunotoxin.

The lens opacity directly decreases visual acuity. To date, neither prophylactic nor pharmacologic treatments have been effective in stopping or decreasing lens opacity. Although extracapsular cataract extraction followed by intraocular lens insertion is considered to be an effective treatment, postoperative proliferation of lens epithelial cells (LECs) disturbs the vision again. In the present study a monoclonal antibody (MAb) directed against bovine LECs was generated. The MAb (XC3-2-B1H9) reacted specifically with the cell membrane of LECs. No other tissues in bovine eyes, such as the cornea, iris, ciliary body, choroid, retina, or the sclera, or any other major bovine organs, such as lung, liver, spleen, and kidney, were associated. The specificity of XC3-2-B1H9 was confirmed both by immunohistochemistry and by immunoblotting. This MAb cross-reacts with dog LECs. Based on immunoblot analysis, XC3-2-B1H9 recognizes the band with a Mr of about 160 kD of the water-insoluble fraction of LECs. The subclass of these MAbs is IgG1 as determined by immunodiffusion. This MAb has the capacity to act as a component of an immunotoxin to target the LECs.

Search for MHC-associated genes in human: five new genes centromeric to HLA-DP with yet unknown functions.

Taking advantage of five mouse genomic or cDNA probes [KE5(probe 14), KE4 (probe 11), KE3 (probe 7), KE2 (probe 5), and SET] mapped on the H-2K region in mouse, we have identified and localized homologues of these five genes in the human major histocompatibility complex region (HKE5, HKE4, HKE3, HKE2, and HSET, respectively). Cosmid cloning and pulsed field gel electrophoresis analyses indicated that a human homologous gene, HKE5, is located 10 kilobases (kb) centromeric of the alpha 2 (XI) collagen (COL11A2) gene followed by HKE4. HKE3, closely linked to HKE2, is located 170 kb centromeric of HKE4. Furthermore, HSET is located 50 kb centromeric of HKE2. This gene organization outside the DP subregion is completely identical to that of the mouse H-2K region centromeric of I-Pb3, a mouse homologue of the DPB gene, except the lack of genes corresponding to the H-2K and -K2 genes in human.

Differential evolution in chromosomal composition during multiple cycles of subcutaneous and intravenous metastasis.

It has been proven that multiple cycles of metastasis can improve the metastatic potential and homing specificity of a tumor cell population. In the present study, verification of genetic alterations during changes in metastatic behavior was done by analyzing the chromosome composition of a methylcholanthrene induced murine fibrosarcoma, 3AM during multiple cycles of subcutaneous (SC) and intravenous (IV) metastasis. After 10 cycles of SC metastasis, a cell type, 7B, with a small t(19;19)(A;A) metacentric marker chromosome was enriched from 4% in the original population to 90% in FIOR. However, when the tumor cells were injected IV rather than SC, no enrichment of the 7B cell type was observed. Instead, a cell type AX with a large t(14;19)(E5;A) acrocentric marker chromosome was enriched from 1% in the parental population to 76% in F1OIV after 10 cycles of IV metastasis. The polyploid dominant FIOIV was found to be extremely high in IV metastasis (411 foci/lung) but low in SC metastasis (48 foci/lung). The diploid dominant FIOR appears to be high in both SC (163 foci/lung) and IV (301 foci/lung) metastasis. The data obtained suggest that metastasis will lead to the selection of specific preexisting cell types, and the type of cell selected will depend on the route of metastasis. Furthermore, during metastasis, new cell types may also be produced de novo through chromosomal structural and numerical aberrations.

Expression of a specific protein in spontaneously metastatic fibrosarcoma cell lines and its enhanced synthesis by growth on laminin or fibronectin.

A panel of tumor cell lines and clones was generated by selecting for different metastatic capacity in 3AM fibrosarcoma cells. These cell lines were exposed to substrata of various purified extracellular matrix (ECM) proteins and labeled in culture with [35S]methionine. Following analysis by two-dimensional polyacrylamide gel electrophoresis the profiles of total cellular proteins produced by the cell lines displaying different phenotypes were examined. The presence of a specific protein, p54, was related to the cellular metastatic potential, and the synthesis of p54 was influenced by growth on extracellular matrix components. The amount of p54 was minimal and non-inducible in tumor cell lines exhibiting two different phenotypes: (1) experimentally metastatic (EM) and (2) transformed, non-tumorigenic (NTT) cell types. In contrast, all of the five different cell lines capable of both spontaneous and experimental metastasis (SEM), produced p54 either constitutively or through induction by growth on ECM protein substrata of either laminin of fibronectin, but not collagen type IV. These data suggest that the p54 protein may be a unique biochemical marker associated with spontaneous metastatic cell types.

Mutations in human lymphocytes commonly involve gene duplication and resemble those seen in cancer cels.

Mutations in human lymphocytes are commonly due to gene deletion. To investigate the mechanism of deletion for autosomal genes, we immunoselected lymphocytes mutated at the HLA-A locus and cloned them for molecular analysis. Of 36 mutant clones that showed deletion of the selected HLA-A allele, 8 had resulted from a simple gene deletion, whereas 28 had resulted from a more complex mutational event involving reduplication of the nonselected HLA-A allele as indicated by hybridization intensity on Southern blots. In 3 of the 28 clones, retention of heterozygosity at the HLA-B locus indicated that the reduplication was due to recombination between the two chromosomes 6; but in the remaining 25 clones, distinction could not be made between recombination and chromosome reduplication. The results indicate that mutations in normal somatic cells frequently result in hemizygosity or homozygosity at gene loci and, thereby, resemble the mutations thought to be important in the etiology of various forms of cancer.

Mutations in human lymphocytes studied by an HLA selection system.

Human lymphocytes mutated at the HLA-A2 or HLA-A3 alleles were enumerated and studied by primary selection using antibody and complement, followed by limiting dilution cloning and secondary selection using immunofluorescence or antibody and complement. The geometric mean frequency of in vivo mutant lymphocytes was 3.08 X 10(-5) for the HLA-A2 allele and 4.68 X 10(-6) for the HLA-A3 allele. Mutagenesis by X-radiation or mitomycin produced a dose-related increase in mutant frequency. HLA-B phenotyping and Southern Analysis of the HLA-A gene suggested that mutation was frequently due to gene deletion, which was often substantial.