Construction, purification, and functional incorporation on tumor cells of glycolipid-anchored human B7-1 (CD80).

To generate a potent cell-mediated immune response, at least two signals are required by T cells. One is engagement of the T-cell receptor with peptide-bearing major histocompatibility complex molecules. The other signal can be delivered by various molecules on the antigen-presenting cell, such as B7-1 (CD80). Many tumor cells escape immune recognition by failing to express these costimulatory molecules. Transfection of the B7 gene into some murine tumor cells allows for immune recognition and subsequent rejection of the parental tumor. We have studied an alternative approach for the introduction of B7-1 onto the surface of tumor cells. This method involves purified glycosyl-phosphatidylinositol (GPI)-anchored proteins which can spontaneously incorporate their lipid tail into cell membranes. We have created and purified a GPI-anchored B7-1 molecule (called GPI-B7) which is able to bind its cognate ligand, CD28, and incorporate itself into tumor cell membranes after a short incubation. Tumor cells that have been reconstituted with GPI-B7 can provide the costimulatory signal needed to stimulate T cells. These findings suggest an approach for the introduction of new proteins onto cell membranes to create an effective tumor vaccine for potential use in human immunotherapy.

Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.

Human anti-idiotypic antibodies induced a humoral and cellular immune response against a colorectal carcinoma-associated antigen in patients.

Induction of immunity against antigens expressed on tumor cells might prevent or delay recurrence of the disease. Six patients operated on for colorectal carcinoma were immunized with human monoclonal anti-idiotypic antibodies (h-Ab2) against the mouse 17-1A anti-colon carcinoma antibody, mimicking a nominal antigen (GA733-2). All patients developed a long-lasting T-cell immunity against the extracellular domain of GA733-2 (GA733-2E) (produced in a baculovirus system) and h-Ab2. This was shown in vitro by specific cell proliferation (DNA-synthesis) assay as well as by interleukin 2 and interferon gamma production and in vivo by the delayed-type hypersensitivity reaction. Five patients mounted a specific humoral response (IgG) against the tumor antigen GA733-2E (ELISA) and tumor cells expressing GA733-2. Epitope mapping using 23 overlapping peptides of GA733-2E revealed that the B-cell epitope was localized close to the N terminus of GA733-2. Binding of the antibodies to the tumor antigen and to one 18-aa peptide was inhibited by h-Ab2, indicating that the antibodies were able to bind to the antigen as well as to h-Ab2. The results suggest that our h-Ab2 might be able to induce an anti-tumor immunity which may control the growth of tumor cells in vivo.

Mutually compensatory mutations during evolution of the tetramerization domain of tumor suppressor p53 lead to impaired hetero-oligomerization

We have measured the stability and stoichiometry of variants of the human p53 tetramerization domain to assess the effects of mutation on homo- and hetero-oligomerization. The residues chosen for mutation were those in the hydrophobic core that we had previously found to be critical for its stability but are not conserved in human p73 or p51 or in p53-related proteins from invertebrates or vertebrates. The mutations introduced were either single natural mutations or combinations of mutations present in p53-like proteins from different species. Most of the mutations were substantially destabilizing when introduced singly. The introduction of multiple mutations led to two opposite effects: some combinations of mutations that have occurred during the evolution of the hydrophobic core of the domain in p53-like proteins had additive destabilizing effects, whereas other naturally occurring combinations of mutations had little or no net effect on the stability, there being mutually compensating effects of up to 9.5 kcal/mol of tetramer. The triple mutant L332V/F341L/L344I, whose hydrophobic core represents that of the chicken p53 domain, was nearly as stable as the human domain but had impaired hetero-oligomerization with it. Thus, engineering of a functional p53 variant with a reduced capacity to hetero-oligomerize with wild-type human p53 can be achieved without any impairment in the stability and subunit affinity of the engineered homo-oligomer.

Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-γ ligand troglitazone in patients with liposarcoma

Agonist ligands for the nuclear receptor peroxisome proliferator-activated receptor-γ have been shown to induce terminal differentiation of normal preadipocytes and human liposarcoma cells in vitro. Because the differentiation status of liposarcoma is predictive of clinical outcomes, modulation of the differentiation status of a tumor may favorably impact clinical behavior. We have conducted a clinical trial for treatment of patients with advanced liposarcoma by using the peroxisome proliferator-activated receptor-γ ligand troglitazone, in which extensive correlative laboratory studies of tumor differentiation were performed. We report here the results of three patients with intermediate to high-grade liposarcomas in whom troglitazone administration induced histologic and biochemical differentiation in vivo. Biopsies of tumors from each of these patients while on troglitazone demonstrated histologic evidence of extensive lipid accumulation by tumor cells and substantial increases in NMR-detectable tumor triglycerides compared with pretreatment biopsies. In addition, expression of several mRNA transcripts characteristic of differentiation in the adipocyte lineage was induced. There was also a marked reduction in immunohistochemical expression of Ki-67, a marker of cell proliferation. Together, these data indicate that terminal adipocytic differentiation was induced in these malignant tumors by troglitazone. These results indicate that lineage-appropriate differentiation can be induced pharmacologically in a human solid tumor.

HLA-G expression in melanoma: A way for tumor cells to escape from immunosurveillance

Considering the well established role of nonclassical HLA-G class I molecules in inhibiting natural killer (NK) cell function, the consequence of abnormal HLA-G expression in malignant cells should be the escape of tumors from immunosurveillance. To examine this hypothesis, we analyzed HLA-G expression and NK sensitivity in human malignant melanoma cells. Our analysis of three melanoma cell lines and ex vivo biopsy demonstrated that (i) IGR and M74 human melanoma cell lines exhibit a high level of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns, (ii) a higher level of HLA-G transcription ex vivo is detected in a skin melanoma metastasis biopsy compared with a healthy skin fragment from the same individual, and (iii) HLA-G protein isoforms other than membrane-bound HLA-G1 protect IGR from NK lysis. It thus appears of critical importance to consider the specific role of HLA-G expression in tumors in the design of future cancer immunotherapies.

Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment

Novel anti-neoplastic agents such as gene targeting vectors and encapsulated carriers are quite large (approximately 100–300 nm in diameter). An understanding of the functional size and physiological regulation of transvascular pathways is necessary to optimize delivery of these agents. Here we analyze the functional limits of transvascular transport and its modulation by the microenvironment. One human and five murine tumors including mammary and colorectal carcinomas, hepatoma, glioma, and sarcoma were implanted in the dorsal skin-fold chamber or cranial window, and the pore cutoff size, a functional measure of transvascular gap size, was determined. The microenvironment was modulated: (i) spatially, by growing tumors in subcutaneous or cranial locations and (ii) temporally, by inducing vascular regression in hormone-dependent tumors. Tumors grown subcutaneously exhibited a characteristic pore cutoff size ranging from 200 nm to 1.2 μm. This pore cutoff size was reduced in tumors grown in the cranium or in regressing tumors after hormone withdrawal. Vessels induced in basic fibroblast growth factor-containing gels had a pore cutoff size of 200 nm. Albumin permeability was independent of pore cutoff size. These results have three major implications for the delivery of therapeutic agents: (i) delivery may be less efficient in cranial tumors than in subcutaneous tumors, (ii) delivery may be reduced during tumor regression induced by hormonal ablation, and (iii) permeability to a molecule is independent of pore cutoff size as long as the diameter of the molecule is much less than the pore diameter.

An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Non-p53 p53RE binding protein, a human transcription factor functionally analogous to P53

The transactivation activity of the p53 tumor suppressor protein is critical for regulating cell growth and apoptosis. We describe the identification of a transcription factor that is functionally similar to p53 and contains the same DNA binding and transcription activities specific for the p53 responsive DNA element (p53RE). This protein was highly purified through chromatography from HeLa cell extracts. The purified protein was able to bind specifically to the p53RE derived from a p21waf1 promoter and to stimulate p53RE-dependent transcription but not basal transcription in vitro. Its DNA-binding activity was inhibited by the wild type but not mutant p53RE-containing DNA oligomers. Also, this p53RE-binding activity was found in human p53 null Saos-2 osteosarcoma and H1299 small cell lung carcinoma cells. Interestingly, this activity exhibited a p53RE sequence preference that was distinct from the p53 protein. The activity is neither p53 nor p73, because anti-p53 or anti-73 antibodies were unable to detect this purified protein nor were the antibodies able to alter the p53-like activity, the p53RE-protein complex. These results demonstrate that, besides p73, an additional p53-like protein exists in cells, which is named NBP for non-p53, p53RE binding protein.

Computerized polymorphic marker identification: Experimental validation and a predicted human polymorphism catalog

A computational system for the prediction of polymorphic loci directly and efficiently from human genomic sequence was developed and verified. A suite of programs, collectively called pompous (polymorphic marker prediction of ubiquitous simple sequences) detects tandem repeats ranging from dinucleotides up to 250 mers, scores them according to predicted level of polymorphism, and designs appropriate flanking primers for PCR amplification. This approach was validated on an approximately 750-kilobase region of human chromosome 3p21.3, involved in lung and breast carcinoma homozygous deletions. Target DNA from 36 paired B lymphoblastoid and lung cancer lines was amplified and allelotyped for 33 loci predicted by pompous to be variable in repeat size. We found that among those 36 predominately Caucasian individuals 22 of the 33 (67%) predicted loci were polymorphic with an average heterozygosity of 0.42. Allele loss in this region was found in 27/36 (75%) of the tumor lines using these markers. pompous provides the genetic researcher with an additional tool for the rapid and efficient identification of polymorphic markers, and through a World Wide Web site, investigators can use pompous to identify polymorphic markers for their research. A catalog of 13,261 potential polymorphic markers and associated primer sets has been created from the analysis of 141,779,504 base pairs of human genomic sequence in GenBank. This data is available on our Web site (pompous.swmed.edu) and will be updated periodically as GenBank is expanded and algorithm accuracy is improved.

Demonstration of an interferon γ-dependent tumor surveillance system in immunocompetent mice

This study demonstrates that endogenously produced interferon γ (IFN-γ) forms the basis of a tumor surveillance system that controls development of both chemically induced and spontaneously arising tumors in mice. Compared with wild-type mice, mice lacking sensitivity to either IFN-γ (i.e., IFN-γ receptor-deficient mice) or all IFN family members (i.e., Stat1-deficient mice) developed tumors more rapidly and with greater frequency when challenged with different doses of the chemical carcinogen methylcholanthrene. In addition, IFN-γ-insensitive mice developed tumors more rapidly than wild-type mice when bred onto a background deficient in the p53 tumor-suppressor gene. IFN-γ-insensitive p53−/− mice also developed a broader spectrum of tumors compared with mice lacking p53 alone. Using tumor cells derived from methylcholanthrene-treated IFN-γ-insensitive mice, we found IFN-γ’s actions to be mediated at least partly through its direct effects on the tumor cell leading to enhanced tumor cell immunogenicity. The importance and generality of this system is evidenced by the finding that certain types of human tumors become selectively unresponsive to IFN-γ. Thus, IFN-γ forms the basis of an extrinsic tumor-suppressor mechanism in immunocompetent hosts.

Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30–42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.

The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis

Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents. However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive. In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas. The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors. Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death. Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells. By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression. These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.

Mouse mammary tumor virus/v-Ha-ras transgene-induced mammary tumors exhibit strain-specific allelic loss on mouse chromosome 4

Hybrid mice carrying oncogenic transgenes afford powerful systems for investigating loss of heterozygosity (LOH) in tumors. Here, we apply this approach to a neoplasm of key importance in human medicine: mammary carcinoma. We performed a whole genome search for LOH using the mouse mammary tumor virus/v-Ha-ras mammary carcinoma model in female (FVB/N × Mus musculus castaneus)F1 mice. Mammary tumors developed as expected, as well as a few tumors of a second type (uterine leiomyosarcoma) not previously associated with this transgene. Genotyping of 94 anatomically independent tumors revealed high-frequency LOH (≈38%) for markers on chromosome 4. A marked allelic bias was observed, with M. musculus castaneus alleles almost exclusively being lost. No evidence of genomic imprinting effects was noted. These data point to the presence of a tumor suppressor gene(s) on mouse chromosome 4 involved in mammary carcinogenesis induced by mutant H-ras expression, and for which a significant functional difference may exist between the M. musculus castaneus and FVB/N alleles. Provisional subchromosomal localization of this gene, designated Loh-3, can be made to a distal segment having syntenic correspondence to human chromosome 1p; LOH in this latter region is observed in several human malignancies, including breast cancers. Evidence was also obtained for a possible second locus associated with LOH with less marked allele bias on proximal chromosome 4.

Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor α

Persistent infection with hepatitis B virus (HBV) is a leading cause of human liver disease and is strongly associated with hepatocellular carcinoma, one of the most prevalent forms of human cancer. Apoptosis (programmed cell death) is an important mediator of chronic liver disease caused by HBV infection. It is demonstrated that the HBV HBx protein acutely sensitizes cells to apoptotic killing when expressed during viral replication in cultured cells and in transfected cells independently of other HBV genes. Cells that were resistant to apoptotic killing by high doses of tumor necrosis factor α (TNFα), a cytokine associated with liver damage during HBV infection, were made sensitive to very low doses of TNFα by HBx. HBx induced apoptosis by prolonged stimulation of N-Myc and the stress-mediated mitogen-activated-protein kinase kinase 1 (MEKK1) pathway but not by up-regulating TNF receptors. Cell killing was blocked by inhibiting HBx stimulation of N-Myc or mitogen-activated-protein kinase kinase 1 using dominant-interfering forms or by retargeting HBx from the cytoplasm to the nucleus, which prevents HBx activation of cytoplasmic signal transduction cascades. Treatment of cells with a mitogenic growth factor produced by many virus-induced tumors impaired induction of apoptosis by HBx and TNFα. These results indicate that HBx might be involved in HBV pathogenesis (liver disease) during virus infection and that enhanced apoptotic killing by HBx and TNFα might select for neoplastic hepatocytes that survive by synthesizing mitogenic growth factors.