Cell configuration and adhesive properties of metastasizing and non-metastasizing BSp73 rat adenocarcinoma cells

The pattern of cell substrate interaction, the cell surface composition and the organization of cytoskeletal elements was studied in tumour cell variants of the BSp73 rat adenocarcinoma displaying different metastatic capabilities and cell configuration. The non-metastasizing AS variant cells adhered to the substrate and spread via vinculin-containing focal contacts. These cells also synthesized, secreted and assembled fibronectin at the pericellular area. The metastasizing ASML variant cells adhered to the substrate at a slower rate via thick cytoplasmic protrusions, but were removed from the substrate by trypsin-EDTA slower than the non-metastasizing AS variant cells. The ASML cells also synthesized very low levels of both vinculin and fibronectin, displayed a diffuse pattern of actin and tubulin organization, and were unable to spread on the substrate. Spreading could not be induced in the ASML cells by seeding the cells on an extracellular matrix derived from bovine corneal endothelial cells or on concanavalin A (conA)-coated substrates, or by the addition of db-cAMP to the medium. The metastasizing cells expressed a unique and abundant cell surface glycoprotein of Mr 170 000 which was also shed into the growth medium. The relationships among the adhesive properties, the organization of cell surface components and of the cytoskeleton in the tumour cell variants, and the expression of their metastatic phenotype is discussed.     

Differences in the distribution of proviruses in low- and highly- metastatic variants of hamster cells, transformed by Rous sarcoma virus

The structure of provirus in Syrian hamster cells, transformed by Rous sarcoma virus (RSV) varying in metastatic capability in vivo has been analysed. The original cell line and its low metastatic variants contain only one copy of the integrated RSV genome. The DNA of highly metastatic cell lines cloned from the same primary culture, contain an additional copy of provirus. This RSV copy in different highly metastatic variants has a similar integration site.     

Cathepsin B-like proteinase as a marker for metastatic tumor cell variants

Serial transplantation of a spontaneous BDX rat tumor, classified as an anaplastic sarcoma, gives rise to two variants; a rapidly growing nonmetastatic line (AS) and a slowly growing, invasive, and highly metastatic variant (ASML). The availability of two cell lines of the same origin but with markedly differing metastatic potential offers an ideal model for the identification of the cellular properties involved in invasive and/or metastatic behavior. The present work focuses on the pattern of various proteinases in the two tumor cell variants. The findings disclosed one major consistent difference which relates to a cathepsin B-like cysteine proteinase. The metastatic ASML variant manifests exceedingly high intracellular cathepsin B-like activity; in the nonmetastatic AS variant, the activity of this proteinase is significantly lower. Other proteinases, in particular elastase-like, chymotrypsin-like, collagenase-like enzymes and plasminogen activator, showed low, essentially comparable activity patterns. Thus, cathepsin B-like proteinase is a marker enzyme of the metastatic ASML tumor cell variant.     

Deoxyadenosine metabolism and cytotoxicity in cultured mouse T lymphoma cells: a model for immunodeficiency disease.

The inherited absence of either adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with severe immunological impairment. We have developed a cell culture model using a mouse T cell lymphoma to simulate ADA deficiency and to study the relationship between purine salvage enzymes and immune function. 2′-deoxyadenosine triphosphate (deoxyATP) levels have been shown to be greatly elevated in erythrocytes of immunodeficient, ADA-deficient patients, suggesting that deoxyadenosine is the potentially toxic substrate in ADA deficiency. Using a potent ADA inhibitor, we have demonstrated that deoxyadenosine is growth-inhibitory and cytotoxic to S49 cells, and that deoxyATP accumulates in these cells. Cell variants, unable to transport or phosphorylate deoxyadenosine, are much less sensitive to deoxyadenosine, indicating that intracellular phosphorylation of deoxyadenosine is required for the lethal effects.We have partially reversed the cytotoxic effects of deoxyadenosine with deoxycytidine in wild-type cells, but we cannot show any reversal in cell lines lacking deoxycytidine kinase. Adenosine (ado) kinase-deficient cells are extremely resistant to deoxyadenosine in the presence of deoxycytidine. This deoxycytidine reversal of deoxyadenosine toxicity is consistent with an inhibition of ribonucleotide reductase by deoxyATP, and we have shown that incubation of S49 cells with deoxyadenosine markedly reduces intracellular levels of deoxyCTP, deoxyGTP and TTP.Kinetics data in wild-type cells and in cell variants are consistent with the presence of two deoxyadenosine-phosphorylating activities — one associated with ado kinase and another associated with deoxycytidine kinase.The S49 cells appear to be a valid model for the simulation of ADA deficiency in cell culture, and from our results, we can suggest administration of deoxycytidine as a pharmacological regimen to circumvent the clinicopathologic symptoms in ADA deficiency.     

Cell surface sialylation and tumor metastasis. Metastatic potential of B16 melanoma variants correlates with their relative numbers of specific penultimate oligosaccharide structures

Numerous investigations suggest that cell surface glycoconjugates, and in particular sialic acids, are directly involved in determining the metastatic phenotype. To further evaluate this hypothesis, we have used a variety of techniques to probe the cell surfaces of several metastatic variants of the murine B16 melanoma that were selected for experimental lung-colonizing ability (Fidler, I. (1973) Nature 242, 148-149) or for their ability to spontaneously metastasize from the site of a subcutaneous injection (Stackpole, C. W., Alterman, A. L., and Fornabaio, D. M. (1985) Invasion & Metastasis 5, 125-142). Using a highly sensitive high performance liquid chromatography sialic acid assay in conjunction with Vibrio cholerae sialidase, we find that none of these metastatic variants differ significantly in their overall levels of cell surface sialic acid. Using highly purified, linkage-specific sialyltransferases, in conjunction with specific glycosidases, to probe the cell surface saccharide topography of specific penultimate oligosaccharides, we also find no significant differences between the efficient lung-colonizing variant, B16-F10 and the poorly-colonizing B16-F1 or B16-Flr variants. In contrast, the spontaneously metastatic variants examined contain substantially different levels of specific penultimate sialylation sites. The tumorigenic but nonmetastatic B16-LM3/G3.26 variant contains 4-fold more penultimate Gal beta 1-3GalNAc sialylation sites than the tumorigenic and highly metastatic B16-LM3/G3.12 variant when CMP[3H]NeuAc and the alpha 2-3Gal beta 1-3GalNAc sialyltransferase are used to probe the melanoma cell surfaces. Several prominent glycoconjugates of apparent Mr 43,000, 40,000, and 30,000 are especially evident upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the nonmetastatic cells. The nonmetastatic variant also contains 2-fold more Gal beta 1-4GlcNAc sialylation sites than the metastatic variant when the alpha 2-6Gal beta 1-4GlcNAc sialyltransferase is used as a cell surface probe. In this case, glycoconjugates of apparent Mr 74,000, 45,000, and 43,000 are more prominently observed on the cell surfaces of the nonmetastatic variant. These data indicate that the differences in lung-colonizing abilities of B16 melanoma metastatic variants do not correlate with the numbers or sialylation states of specific penultimate oligosaccharide structures on their surfaces. However, the relative levels of specific penultimate saccharide structures do correlate with the ability of the cells to undergo spontaneous metastasis from a subcutaneous tumor.     

Immunoselection of tumor variants resistant to antibody-mediated cytotoxicity

Summary The immunological characteristics of two series of metastatic variants of restricted genetic origin were related to their lung-colony-forming potential. A series of metastatic variants was isolated from a tumor-cell population in which heterogeneity appeared following short-term in vivo passage, while a second series of variants were immunoselected in vitro for resistance to antibody-complement-mediated cell lysis. In the case of the first series, the sensitivity of the individual variants to cell-mediated and antibody-complement-mediated cytotoxicity appeared to be partly determined by the number and rate of loss of antibody-binding sites on the cell surface. These characteristics also correlated with the efficiency of experimental metastasis in the animal. We compared the results reported herein with our previous studies of nonimmune parameters for this series of metastatic variants, and we conclude that immunological differences can be important to the efficiency of lung-colony formation. However, in the case of the second series of variants, despite selection in vitro for resistance to antibody-complement-mediated cell lysis, the behavior of these variants in the lung colony assay could not be predicted by the immunologic parameters examined. The abbreviations used, FBS, fetal bovine serum; MEME, Eagle's minimum essential medium; TBSS, Tyrode's balanced salt solution; CMF, Ca²⁺ and Mg²⁺-free Tyrode's balanced salt solution; EGTA, ethyleneglycol-bis-(-amino-ethyl-ether) N,N'-tetra-acetic acid; HEPES, N-2-hydroxy-ethyl-piperazine N 1-2 ethanesulfonic acid; NK, natural killer cell; SDS, sodium dodecyl sulphate; IUDR, iododeoxyuridine; AC, antibody-complementThis study was supported in part by American Cancer Society grants RD-78 and IN-119     

The cell surface sulphydryl content of metastatic variants of B16 murine melanoma

The cell surface sulphydryl content of three metastatic variants of the B16 murine melanoma has been determined using isoelectric equilibrium techniques. The F1 variant, which has no ability for natural metastasis, and the F10 variant with moderate metastatic ability appeared to have no detectable surface thiol groups. The variant BL6, which shows a high degree of natural metastasis, possessed surface thiol groups. The variants were found to be heterogeneous in isoelectric distribution. Three subpopulations were identified based on isoelectric criteria. The size of the pI 5.0 subpopulation appeared to increase with metastatic ability. A proportion of this subpopulation, approximately 4% in the F10 and 10% in the BL6, was found to possess surface thiol groups. In the BL6 line, 10-20% of the pI 4.6 subpopulation possessed surface thiol groups. The surface negative charge density of the cell types showed no correlation with their natural metastatic ability.     

Mutational signatures of de-differentiation in functional non-coding regions of melanoma genomes

Much emphasis has been placed on the identification, functional characterization, and therapeutic potential of somatic variants in tumor genomes. However, the majority of somatic variants lie outside coding regions and their role in cancer progression remains to be determined. In order to establish a system to test the functional importance of non-coding somatic variants in cancer, we created a low-passage cell culture of a metastatic melanoma tumor sample. As a foundation for interpreting functional assays, we performed whole-genome sequencing and analysis of this cell culture, the metastatic tumor from which it was derived, and the patient-matched normal genomes. When comparing somatic mutations identified in the cell culture and tissue genomes, we observe concordance at the majority of single nucleotide variants, whereas copy number changes are more variable. To understand the functional impact of non-coding somatic variation, we leveraged functional data generated by the ENCODE Project Consortium. We analyzed regulatory regions derived from multiple different cell types and found that melanocyte-specific regions are among the most depleted for somatic mutation accumulation. Significant depletion in other cell types suggests the metastatic melanoma cells de-differentiated to a more basal regulatory state. Experimental identification of genome-wide regulatory sites in two different melanoma samples supports this observation. Together, these results show that mutation accumulation in metastatic melanoma is nonrandom across the genome and that a de-differentiated regulatory architecture is common among different samples. Our findings enable identification of the underlying genetic components of melanoma and define the differences between a tissue-derived tumor sample and the cell culture created from it. Such information helps establish a broader mechanistic understanding of the linkage between non-coding genomic variations and the cellular evolution of cancer.     

Expression of PNA-binding sites on specific glycoproteins by human melanoma cells is associated with a high metastatic potential

Lectin-binding patterns of seven human melanoma clones and variants selected from the same parental cell line and differing in their spontaneous metastatic potential in an animal model were compared by flow cytometry and Scatchard analysis. Human melanoma clones and variants with high and low metastatic potential could be distinguished by their peanut agglutinin (PNA)–binding patterns, but not by their wheat germ agglutinin (WGA)–, Ulex europaeus agglutinin I (UEA I)–, and soybean agglutinin (SBA)–binding patterns. Low metastatatic clones and variants proved to be made up of a single poorly peanut agglutinin–binding cell population (2.20–3.52 × 10⁶ sites/cell, Ka = 2.48–2.75 × 10⁶ M^?1). By contrast, highly metastatic variants were found to be constituted by two cellular subpopulations, exhibiting respectively a moderate (2.62–3.72 × 10⁶ sites/cell) and a high peanut agglutinin staining (17.68–18.76 × 10⁶ sites/cell). One highly metastatic clone was found to be homogeneously constituted by a single population of cells strongly binding this lectin (18.86 × 10⁶ sites/cell) with an association constant of 4.06 × 10⁶ M^?1. Using an EPICS V cytometer, these two subpopulations were sorted from a highly metastatic variant and tested for their metastatic abilities: cells with high PNA binding generated a higher frequency of metastases than did moderately PNA-binding cells. Following treatment with Vibrio cholerae neuraminidase, all cells from all variants and clones were brightly labeled by PNA, collecting in a single peak with similar fluorescence intensities. Electrophoresis of total cellular proteins and subsequent detection with labeled PNA on Western blots show two major PNA-reactive glycoproteins with apparent molecular weights of 140 and 110 kDa (MAGP1 and MAGP2), expressed only in highly metastatic cells, but which can be strongly labeled by PNA in slightly metastatic cells following a treatment with neuraminidase. These results provide evidence that the expression of terminal galactose (β1–3)N-acetyl galactosamine structures, positioned on MAGP1 and MAGP2 glycoproteins, is associated with the metastatic potential of human melanoma cells.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Metastatic tumor cell variants with increased resistance to infection by Semliki Forest virus

Semliki Forest virus (SFV) is an interesting virus for cell interaction studies because it binds directly to the cells' major histocompatibility antigens. We used this reagent to study the expression and functional properties of H-2 molecules on murine tumor lines that are closely related but differ greatly in metastatic capacity. Tumor cell variants with high metastatic capacity showed an increased resistance to virus infection, an effect that was selective for SFV. Although the high metastatic tumor lines did not express less H-2 antigens than the low metastatic ones, they bound much less of the SFV viral glycoproteins.     

Osteopontin Splice Variants Differentially Exert Clinicopathological Features and Biological Functions in Gastric Cancer

Purpose: Gastric cancer (GC) remains a leading cause of death worldwide, and an elevated expression of osteopontin (OPN) may correlate with its poor survival. Alternative splicing of OPN can result in three isoforms, OPN-a, OPN-b and OPN-c. The aim of our current study is to examine the expression pattern and biological functions of OPN splice variants in GC.Methods: Firstly, we evaluated the expression of OPN splice variants in 7 gastric cell lines, 101 pairs of GC tissues and their adjacent non-tumor tissues by Quantative real-time PCR (QT-PCR). Gain-of-function experiments were subsequently performed to determine their diverse roles in malignant behaviors of GC. Besides, their differential effects on the regulation of crucial downstream molecules were further explored in the anti-apoptotic and pro-metastatic process.Results: We found that OPN-b is the dominant kind of OPN isoform in GC cell lines. Although the expression levels of three variants were all elevated in GC tissues, increased OPN-b or OPN-c expression could correlate with clinicopathological features. Functional analyses further showed that OPN-b most strongly promoted GC cell survival possibly by regulation of Bcl-2 family proteins and CD44v expressions. Moreover, OPN-c most effectively stimulated GC metastatic activity by increasing secretion of MMP-2, uPa, and IL-8.Conclusions: Our results suggest that OPN splice variants differentially exert clinicopathological features and biological functions in GC. Therefore, focusing on specific OPN isoform could be a novel direction for developing diagnostic and therapeutic approaches in GC.     

AFM-based dual nano-mechanical phenotypes for cancer metastasis

An enhanced mechanical compliance is considered to be a mechanical indicator for metastatic cancer cells. Our study using atomic force microscopy (AFM) revealed that breast cancer cells agreed well with this hypothesis. However, prostate cancer cells displayed a reverse correlation; less metastatic prostate cancer cells were more mechanically compliant. Two-dimensional AFM force spectroscopy was performed to characterize dual mechanical properties—the cell–substrate adhesion as well as the mechanical compliance. Interestingly, prostate cancer cells displayed a strong positive correlation between the cell–substrate adhesion and metastatic potential. However, there was no clearly observable correlation between the cell–substrate adhesion and the metastatic potential despite variations in mechanical compliance of breast cancer cells. These results suggest that the correlation between the dual mechanical signatures and metastatic potential be uniquely identified for cancer cells originating from different organs. We postulate that this correlation could reveal which step of cancer progression is favorable in terms of physical interaction between cancer cells and micro-environments. We expect that based on the “seed and soil hypothesis”, the identification of the dual mechanical phenotypes, could provide a new insight for understanding how a dominant metastatic site is determined for cancer cells originating from specific organs.     

Transforming potential of alternatively spliced variants of fibroblast growth factor receptor 2 in human mammary epithelial cells

A breast cancer cell line developed in our laboratory (SUM-52PE) has a 12-fold amplification and high-level overexpression of the oncogene fibroblast growth factor receptor 2 (FGFR2). Previously, nine different alternatively spliced FGFR2 variants were isolated from this cell line. Overexpression of two variants that differ only in their carboxyl termini (C1 and C3) has been successfully accomplished in the immortalized human mammary epithelial cell line H16N2. FGFR2 expression led to the activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling cascades. Phosphorylation of the adapter protein FGF receptor substrate 2 is much more robust in the cells expressing the C3 variant of FGFR2 compared with the C1 variant. H16N2 cells expressing the full-length FGFR2 with the C1 or C3 carboxyl terminus were tested for their ability to grow under epidermal growth factor (EGF)-independent conditions, in soft agar, and for their ability to invade naturally occurring basement membranes and compared with the parental SUM-52PE cell line. All three cell lines grew under EGF-independent conditions and all were inhibited by the FGFR family specific inhibitor PD173074. The full-length FGFR2-C1 and FGFR2-C3 variants grew robustly in soft agar similar to the parental cell line SUM-52PE. However, cells expressing the C3 variant formed large colonies in agar in both insulin-free and EGF-free medium, whereas the cells expressing the C1 variant required insulin for growth. Soft agar growth was also inhibited by PD173074. Because SUM-52PE was developed from a metastatic breast carcinoma, the FGFR2-overexpressing cell lines were assessed for their ability to invade sea urchin embryo cell membranes. H16N2 cells expressing the C1 carboxyl terminus failed to invade sea urchin embryo cell membranes. By contrast, FGFR2-C3-expressing cells were as invasive as the SUM-52 breast cancer cells and erbB-2-overexpressing H16N2 cells. These results indicate that FGFR2 is a transforming oncogene in human mammary epithelial cells when expressed to levels similar to that found in breast cancer cells with FGFR2 gene amplification. Furthermore, the results suggest that different splice variants have differing transforming activities and that signaling from variants expressing the C3 carboxyl terminus results in more autonomous signaling, cell growth, and invasion.     

Metastasis-associated alterations in phospholipids and fatty acids of human prostatic adenocarcinoma cell lines.

Metastatic variants of human prostatic adenocarcinoma cell lines (DU-145, LNCaP, and ND-1) were studied by using soft agar colony forming efficiency, nude mice tumorigenicity, in vitro invasion assay, and type IV collagenase assay. The DU-145 and ND-1 cell line showed higher metastatic potential than LNCaP. Lipids from DU-145, ND-1, and LNCaP cells were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. The major lipids were phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, fatty acids, and cholesterol. The sphingomyelin level was significantly higher in highly metastatic cells (DU-145 and ND-1) compared with the lower metastatic variant (LNCaP). The increase in the synthetic pathway and decrease in degradation pathway of sphingomyelin in microsomal fractions was sufficient to account for the measured increase in sphingomyelin in DU-145 cells compared with LNCaP cells. The major fatty acids of these lipids were palmitic (16:0), stearic (18:0), oelic (18:1), and arachidonic acid (20:4). The arachidonic acid level was significantly decreased in DU-145 and ND-1 compared with LNCaP cells. Electron microscopic studies showed no significant changes in the morphology of DU-145, ND-1, and LNCaP cells. The results of these investigations demonstrate for the first time that sphingomyelin and arachidonic acid contents are different in high and low metastatic variants of human prostatic adenocarcinoma cell lines.     

Proportion of antigenic variants induced by in vitro UV irradiation differs in clones derived from a single tumor

The purpose of this study was to examine the capacity of different clones derived from the same tumor to generate highly antigenic cells after in vitro exposure to UV radiation. Cells from the metastatic murine melanoma K1735 and clones of K1735 differing in metastatic potential were exposed to UV radiation in vitro, cloned, and tested for antigenic properties in vivo. Approximately half of the clones isolated after UV irradiation of parental K1735 melanoma cells were highly antigenic (five of nine). Similar treatment of cells of a nonmetastatic clone of K1735 generated clones that were all antigenic (nine of nine). In contrast, only one of nine clones derived from UV-irradiated cells of a highly metastatic clone of K1735 were antigenic. Clones derived from unirradiated cultures were not antigenic variants. The increased antigenicity of cells derived from UV-irradiated cultures did not correlate with an increase in expression of cell surface class I major histocompatibility complex antigens. These results demonstrate that the frequency of antigenic variant production after UV irradiation is an intrinsic property of the particular cell line used, and that even cloned cell lines derived from a single tumor differ in their ability to generate antigenic variants after UV irradiation. In addition, they indicate that the increased antigenicity is not necessarily due to a UV-induced increase in expression of cell surface class I histocompatibility antigens.     

Expression of CD44 Splice Variants During Lymphocyte Activation and Tumor Progression

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

Expression of CD44 Splice Variants During Lymphocyte Activation and Tumor Progression

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

Protein kinase C and its substrates in tumor promoter-sensitive and -resistant cells

Calcium- and phospholipid-dependent protein kinase C activity and substrates were characterized in cell lysates of preneoplastic JB6 cells, a model system of genetic variants for sensitivity to tumor promoter-induced neoplastic transformation. Protein kinase C activity was similar for sensitive and resistant variants, as measured by calcium- and phospholipid-dependent phosphorylation of an exogenous substrate (histone HIII). Of 13 endogenous protein kinase C substrates, identified by labeling proteins with [gamma-32P] ATP, at least two (80 and 23 kDa) are potential candidates for mediating events on the pathway for promotion of transformation. 32P incorporation into the 80-kDa protein kinase C substrate was stimulated by tetradecanoylphorbol acetate and correlated with phenotype: the highest incorporation was found in promotion-insensitive cells, an intermediate level in promotion-sensitive cells and the lowest in the transformed cells. The phosphorylation of an 80-kDa protein, found by labeling intact cells in monolayer growth with [32P]orthophosphate, was also stimulated by tetradecanoylphorbol acetate and correlated inversely with phenotype. The 80 kDa protein kinase C substrate from cell lysates and the 80-kDa phosphoprotein from intact cells appear to be identical, as indicated by peptide mapping with protease V8 from Staphylococcus aureus. This finding suggests that the 80-kDa substrate is relevant to promoter-induced signal transduction in the intact cell. The 23-kDa protein kinase C substrate exhibited a band shift in sodium dodecyl sulfate gels in response to another transformation promoter in JB6 cells, the calcium analog, lanthanum (Smith, B. M., Gindhart, T. D., and Colburn, N. H. (1986) Carcinogenesis 7, 1949-1956). In summary, there are no unique substrates that distinguish the variants. Quantitative differences in certain substrates or their phosphorylation may, however, account for the difference in promotion sensitivity among the variants.     

The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding

Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding.