Tissue factor promotes melanoma metastasis by a pathway independent of blood coagulation.

Several studies have established a link between blood coagulation and cancer, and more specifically between tissue factor (TF), a transmembrane protein involved in initiating blood coagulation, and tumor metastasis. In the study reported here, a murine model of human melanoma metastasis was used for two experiments. (i) The first experiment was designed to test the effect of varying the level of TF expression in human melanoma cells on their metastatic potential. Two matched sets of cloned human melanoma lines, one expressing a high level and the other a low level of the normal human TF molecule, were generated by retroviral-mediated transfections of a nonmetastatic parental line. The metastatic potential of the two sets of transfected lines was compared by injecting the tumor cells into the tail vein of severe combined immunodeficiency (SCID) mice and later examining the lungs and other tissues for tumor development. Metastatic tumors were detected in 86% of the mice injected with the high-TF lines and in 5% of the mice injected with the low-TF lines, indicating that a high TF level promotes metastasis of human melanoma in the SCID mouse model. This TF effect on metastasis occurs with i.v.-injected melanoma cells but does not occur with primary tumors formed from s.c.-injected melanoma cells, suggesting that TF acts at a late stage of metastasis, after tumor cells have escaped from the primary site and entered the blood. (ii) The second experiment was designed to analyze the mechanism by which TF promotes melanoma metastasis. The procedure involved testing the effect on metastasis of mutations in either the extracellular or cytoplasmic domains of the transmembrane TF molecule. The extracellular mutations introduced two amino acid substitutions that inhibited initiation by TF of the blood-coagulation cascade; the cytoplasmic mutation deleted most of the cytoplasmic domain without impairing the coagulation function of TF. Several human melanoma lines expressing high levels of either of the two mutant TF molecules were generated by retroviral-mediated transfection of the corresponding TF cDNA into the nonmetastatic parental melanoma line, and the metastatic potential of each transfected line was tested in the SCID mouse model. Metastases occurred in most mice injected with the melanoma lines expressing the extracellular TF mutant but were not detected in most mice injected with the melanoma lines expressing the cytoplasmic TF mutant. Results with the extracellular TF mutant indicate that the metastatic effect of TF in the SCID mouse model does not involve products of the coagulation cascade. Results with the cytoplasmic TF mutant indicate that the cytoplasmic domain of TF is important for the metastatic effect, suggesting that the TF could transduce a melanoma cell signal that promotes metastasis.

Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma

Translocations involving c-myc and an Ig locus have been reported rarely in human multiple myeloma (MM). Using specific fluorescence in situ hybridization probes, we show complex karyotypic abnormalities of the c-myc or L-myc locus in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors. These abnormalities include unusual and complex translocations and insertions that often juxtapose myc with an IgH or IgL locus. For two advanced primary MM tumors, some tumor cells contain a karyotypic abnormality of the c-myc locus, whereas other tumor cells do not, indicating that this karyotypic abnormality of c-myc occurs as a late event. All informative MM cell lines show monoallelic expression of c-myc. For Burkitt's lymphoma and mouse plasmacytoma tumors, balanced translocation that juxtaposes c-myc with one of the Ig loci is an early, invariant event that is mediated by B cell-specific DNA modification mechanisms. By contrast, for MM, dysregulation of c-myc apparently is caused principally by complex genomic rearrangements that occur during late stages of MM progression and do not involve B cell-specific DNA modification mechanisms.

Lack of the DNA repair protein O6-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary human brain tumors.

Exposure to exogenous alkylating agents, particularly N-nitroso compounds, has been associated with increased incidence of primary human brain tumors, while intrinsic risk factors are currently unknown. The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a major defense against the carcinogenicity of N-nitroso compounds and other alkylators. We report here that in 55% (64/117) of cases, histologically normal brain tissue adjacent to primary human brain tumors lacked detectable MGMT activity [methyl excision repair-defective (Mer-) status]. The incidence of Mer- status in normal brain tissue from brain tumor patients was age-dependent, increasing from 21% in children 0.25-19 years of age to 75% in adults over 50. In contrast, Mer- status was found in 12% (5/43) of normal brain specimens from patients operated for conditions other than primary brain tumors and was not age-dependent. The 4.6-fold elevation in incidence of Mer- status in brain tumor patients is highly significant (chi2 = 24; p < or = 0.001). MGMT activity was independent of age in the lymphocytes of brain tumor patients and was present in lymphocytes from six of nine tumor patients whose normal brain specimen was Mer-. DNA polymerase beta, apurinic/apyrimidinic endonuclease, and lactate dehydrogenase activities were present in all specimens tested, including Mer- specimens from brain tumor patients. Our data are consistent with a model of carcinogenesis in human brain in which epigenetically regulated lack of MGMT is a predisposing factor and alkylation-related mutagenesis is a driving force.

Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors.

Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 +/- 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with IL-12 cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional IL-12 gene transfections, could significantly inhibit systemic metastases, resulting in extended survival of test mice. These results suggest that gene gun-mediated in vivo delivery of IL-12 cDNA should be further developed for potential clinical testing as an approach for human cancer gene therapy.

Adaptive mutations in Escherichia coli as a model for the multiple mutational origins of tumors.

The cells in most tumors are found to carry multiple mutations; however, based upon mutation rates determined by fluctuation tests, the frequency of such multiple mutations should be so low that tumors are never detected within human populations. Fluctuation tests, which determine the cell-division-dependent mutation rate per cell generation in growing cells, may not be appropriate for estimating mutation rates in nondividing or very slowly dividing cells. Recent studies of time-dependent, "adaptive" mutations in nondividing populations of microorganisms suggest that similar measurements may be more appropriate to understanding the mutation origins of tumors. Here I use the ebgR and ebgA genes of Escherichia coli to measure adaptive mutation rates where multiple mutations are required for rapid growth. Mutations in either ebgA or ebgR allow very slow growth on lactulose (4-O-beta-D-galactosyl-D-fructose), with doubling times of 3.2 and 17.3 days, respectively. However, when both mutations are present, cells can grow rapidly with doubling times of 2.7 hr. I show that during prolonged (28-day) selection for growth on lactulose, the number of lactulose-utilizing mutants that accumulate is 40,000 times greater than can be accounted for on the basis of mutation rates measured by fluctuation tests, but is entirely consistent with the time-dependent adaptive mutation rates measured under the same conditions of prolonged selection.

RB-mediated suppression of spontaneous multiple neuroendocrine neoplasia and lung metastases in Rb+/− mice

Alterations in pathways mediated by retinoblastoma susceptibility gene (RB) product are among the most common in human cancer. Mice with a single copy of the Rb gene are shown to develop a syndrome of multiple neuroendocrine neoplasia. The earliest Rb-deficient atypical cells were identified in the intermediate and anterior lobes of the pituitary, the thyroid and parathyroid glands, and the adrenal medulla within the first 3 months of postnatal development. These cells form gross tumors with various degrees of malignancy by postnatal day 350. By age of 380 days, 84% of Rb+/− mice exhibited lung metastases from C-cell thyroid carcinomas. Expression of a human RB transgene in the Rb+/− mice suppressed carcinogenesis in all tissues studied. Of particular clinical relevance, the frequency of lung metastases also was reduced to 12% in Rb+/− mice by repeated i.v. administration of lipid-entrapped, polycation-condensed RB complementary DNA. Thus, in spite of long latency periods during which secondary alterations can accumulate, the initial loss of Rb function remains essential for tumor progression in multiple types of neuroendocrine cells. Restoration of RB function in humans may prove an effective general approach to the treatment of RB-deficient disseminated tumors.

Multiple loop structures critical for ligand binding of the integrin α4 subunit in the upper face of the β-propeller mode 1

A non-I-domain integrin, α4β1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of α4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of α4 (which spans repeats 2–5 of the seven N-terminal repeats) with the corresponding regions of α5. Swapping residues 112–131 in repeat 2, or residues 237–247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40–52 in repeat 1, residues 151–164 in repeat 3, or residues 282–288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112–131, 151–164, and 186–191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published β-propeller folding model of the integrin α4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65–72], in which seven four-stranded β-sheets are arranged in a torus around a pseudosymmetric axis. The regions of α4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the β-propeller model, although they are not adjacent in the primary structure.

Syncytiotrophoblastic giant cells in teratocarcinoma-like tumors derived from Parp-disrupted mouse embryonic stem cells

The enzyme poly(ADP-ribose) polymerase (Parp) catalyzes poly(ADP-ribosyl)ation reaction and is involved in DNA repair and cell death induction upon DNA damages. Meanwhile, poly(ADP-ribosyl)ation of chromosome-associated proteins is suggested to be implicated in the regulation of gene expression and cellular differentiation, both of which are important in tumorigenesis. To investigate directly the role of Parp deficiency in tumorigenicity and differentiation of embryonic stem (ES) cells during tumor formation, studies were conducted by using wild-type J1 (Parp+/+) ES cells and Parp+/− and Parp−/− ES clones generated by disrupting Parp exon 1. These ES cells, irrespective of the Parp genotype, produced tumors phenotypically similar to teratocarcinoma when injected s.c. into nude mice. Remarkably, all tumors derived from Parp−/− clones contained syncytiotrophoblastic giant cells (STGCs), which possess single or multiple megalo-nuclei. The STGCs were present within large areas of intratumoral hemorrhage. In contrast, neither STGC nor hemorrhage was observed in tumors of both wild-type J1 cells and Parp+/− clones. Electron microscopic examination showed that the STGCs possess microvilli on the cell surface and contained secretory granules in the cytoplasm. Furthermore, the cytoplasms of STGCs were strongly stained with antibody against mouse prolactin, which could similarly stain trophoblasts in placenta. These morphological and histochemical features indicate that the STGCs in teratocarcinoma-like tumors derived from Parp−/− clones belong to the trophoblast cell lineage. Our findings thus suggest that differentiation of ES cells into STGCs was possibly induced by the lack of Parp during the development of teratocarcinoma.

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging.

Protective anti-tumor immunity induced by vaccination with recombinant adenoviruses encoding multiple tumor-associated cytotoxic T lymphocyte epitopes in a string-of-beads fashion

Vaccines harboring genes that encode functional oncoproteins are intrinsically hazardous, as their application may lead to introduction of these genes into normal cells and thereby to tumorigenesis. On the other hand, oncoproteins are especially attractive targets for immunotherapy of cancer, as their expression is generally required for tumor growth, making the arisal of tumor variants lacking these antigens unlikely. Using murine tumor models, we investigated the efficacy of polyepitope recombinant adenovirus (rAd) vaccines, which encode only the immunogenic T cell epitopes derived from several oncogenes, for the induction of protective anti-tumor immunity. We chose to employ rAd, as these are safe vectors that do not induce the side effects associated with, for example, vaccinia virus vaccines. A single polyepitope rAd was shown to give rise to presentation of both H-2 and human leukocyte antigen-restricted cytotoxic T lymphocyte (CTL) epitopes. Moreover, vaccination with a rAd encoding H-2-restricted CTL epitopes, derived from human adenovirus type 5 early region 1 and human papilloma virus type 16-induced tumors, elicited strong tumor-reactive CTL and protected the vaccinated animals against an otherwise lethal challenge with either of these tumors. The protection induced was superior compared with that obtained by vaccination with irradiated tumor cells. Thus, vaccination with polyepitope rAd is a powerful approach for the induction of protective anti-tumor immunity that allows simultaneous immunization against multiple tumor-associated T cell epitopes, restricted by various major histocompatibility complex haplotypes.

The Ras/Rac1/Cdc42/SEK/JNK/c-Jun Cascade Is a Key Pathway by Which Agonists Stimulate DNA Synthesis in Primary Cultures of Rat Hepatocytes

The ability of signaling via the JNK (c-Jun NH2-terminal kinase)/stress-activated protein kinase cascade to stimulate or inhibit DNA synthesis in primary cultures of adult rat hepatocytes was examined. Treatment of hepatocytes with media containing hyperosmotic glucose (75 mM final), tumor necrosis factor α (TNFα, 1 ng/ml final), and hepatocyte growth factor (HGF, 1 ng/ml final) caused activation of JNK1. Glucose, TNFα, or HGF treatments increased phosphorylation of c-Jun at serine 63 in the transactivation domain and stimulated hepatocyte DNA synthesis. Infection of hepatocytes with poly-l-lysine–coated adenoviruses coupled to constructs to express either dominant negatives Ras N17, Rac1 N17, Cdc42 N17, SEK1−, or JNK1− blunted the abilities of glucose, TNFα, or HGF to increase JNK1 activity, to increase phosphorylation of c-Jun at serine 63, and to stimulate DNA synthesis. Furthermore, infection of hepatocytes by a recombinant adenovirus expressing a dominant-negative c-Jun mutant (TAM67) also blunted the abilities of glucose, TNFα, and HGF to stimulate DNA synthesis. These data demonstrate that multiple agonists stimulate DNA synthesis in primary cultures of hepatocytes via a Ras/Rac1/Cdc42/SEK/JNK/c-Jun pathway. Glucose and HGF treatments reduced glycogen synthase kinase 3 (GSK3) activity and increased c-Jun DNA binding. Co-infection of hepatocytes with recombinant adenoviruses to express dominant- negative forms of PI3 kinase (p110α/p110γ) increased basal GSK3 activity, blocked the abilities of glucose and HGF treatments to inhibit GSK3 activity, and reduced basal c-Jun DNA binding. However, expression of dominant-negative PI3 kinase (p110α/p110γ) neither significantly blunted the abilities of glucose and HGF treatments to increase c-Jun DNA binding, nor inhibited the ability of these agonists to stimulate DNA synthesis. These data suggest that signaling by the JNK/stress-activated protein kinase cascade, rather than by the PI3 kinase cascade, plays the pivotal role in the ability of agonists to stimulate DNA synthesis in primary cultures of rat hepatocytes.

Guanylyl cyclase C is a selective marker for metastatic colorectal tumors in human extraintestinal tissues

Guanylyl cyclase C (GCC) has been detected only in intestinal mucosa and colon carcinoma cells of placental mammals. However, this receptor has been identified in several tissues in marsupials, and its expression has been suggested in tissues other than intestine in placental mammals. Selective expression of GCC by colorectal tumor cells in extraintestinal tissues would permit this receptor to be employed as a selective marker for metastatic disease. Thus, expression of GCC was examined in human tissues and tumors, correlating receptor function with detection by PCR. GCC was detected by ligand binding and catalytic activation in normal intestine and primary and metastatic colorectal tumors, but not in extraintestinal tissues or tumors. Similarly, PCR yielded GCC-specific amplification products with specimens from normal intestine and primary and metastatic colorectal tumors, but not from extraintestinal tissues or tumors. Northern blot analysis employing GCC-specific probes revealed an ≈4-kb transcript, corresponding to recombinant GCC, in normal intestine and primary and metastatic colorectal tumors, but not in extraintestinal tissues. Thus, GCC is selectively expressed in intestine and colorectal tumors in humans and appears to be a relatively specific marker for metastatic cancer cells in normal tissues. Indeed, PCR of GCC detected tumor cells in blood from some patients with Dukes B colorectal cancer and all patients examined with Dukes C and D colorectal cancer, but not in that from normal subjects or patients with Dukes A colon carcinoma or other nonmalignant intestinal pathologies.

Endogenous expression of Müllerian inhibiting substance in early postnatal rat Sertoli cells requires multiple steroidogenic factor-1 and GATA-4-binding sites

Müllerian inhibiting substance (MIS) is a key element required to complete mammalian male sex differentiation. The expression pattern of MIS is tightly regulated in fetal, neonatal, and prepubertal testes and adult ovaries and is well conserved among mammalian species. Although several factors have been shown to be essential to MIS expression, its regulatory mechanisms are not fully understood. We have examined MIS promoter activity in 2-day postnatal primary cultures of rat Sertoli cells that continue to express endogenous MIS mRNA. Using this system, we found that the region between human MIS−269 and −192 is necessary for full MIS promoter activity. We identified by DNase I footprint and electrophoretic mobility-shift analyses a distal steroidogenic factor-1 (SF-1)-binding site that is essential for full promoter activity. Mutational analysis of this new distal SF-1 site and the previously identified proximal SF-1 site showed that both are necessary for transcriptional activation. Moreover, the proximal promoter also contains multiple GATA-4-binding sites that are essential for functional promoter activity. Thus multiple SF-1- and GATA-4-binding sites in the MIS promoter are required for normal tissue-specific and developmental expression of MIS.

Multiple pathways for ultrafast transduction of light energy in the photosynthetic reaction center of Rhodobacter sphaeroides

A pathway of electron transfer is described that operates in the wild-type reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides. The pathway does not involve the excited state of the special pair dimer of bacteriochlorophylls (P*), but instead is driven by the excited state of the monomeric bacteriochlorophyll (BA*) present in the active branch of pigments along which electron transfer occurs. Pump-probe experiments were performed at 77 K on membrane-bound RCs by using different excitation wavelengths, to investigate the formation of the charge separated state P+HA−. In experiments in which P or BA was selectively excited at 880 nm or 796 nm, respectively, the formation of P+HA− was associated with similar time constants of 1.5 ps and 1.7 ps. However, the spectral changes associated with the two time constants are very different. Global analysis of the transient spectra shows that a mixture of P+BA− and P* is formed in parallel from BA* on a subpicosecond time scale. In contrast, excitation of the inactive branch monomeric bacteriochlorophyll (BB) and the high exciton component of P (P+) resulted in electron transfer only after relaxation to P*. The multiple pathways for primary electron transfer in the bacterial RC are discussed with regard to the mechanism of charge separation in the RC of photosystem II from higher plants.

Multiple independent origins of mitochondrial gene order in birds

Mitochondrial genomes of all vertebrate animals analyzed to date have the same 37 genes, whose arrangement in the circular DNA molecule varies only in the relative position of a few genes. This relative conservation suggests that mitochondrial gene order characters have potential utility as phylogenetic markers for higher-level vertebrate taxa. We report discovery of a mitochondrial gene order that has had multiple independent originations within birds, based on sampling of 137 species representing 13 traditionally recognized orders. This provides evidence of parallel evolution in mitochondrial gene order for animals. Our results indicate operation of physical constraints on mitochondrial gene order changes and support models for gene order change based on replication error. Bird mitochondria have a displaced OL (origin of light-strand replication site) as do various other Reptilia taxa prone to gene order changes. Our findings point to the need for broad taxonomic sampling in using mitochondrial gene order for phylogenetic analyses. We found, however, that the alternative mitochondrial gene orders distinguish the two primary groups of songbirds (order Passeriformes), oscines and suboscines, in agreement with other molecular as well as morphological data sets. Thus, although mitochondrial gene order characters appear susceptible to some parallel evolution because of mechanistic constraints, they do hold promise for phylogenetic studies.