Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines

Induction of wild-type p53 in the ECV-304 bladder carcinoma cell line by infection with a p53 recombinant adenovirus (Ad5CMV-p53) resulted in extensive apoptosis and eventual death of nearly all of the cells. As a strategy to determine the molecular events important to p53-mediated apoptosis in these transformed cells, ECV-304 cells were selected for resistance to p53 by repeated infections with Ad5CMV-p53. We compared the expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells by reverse transcription–PCR, Northern blotting, and DNA microarray analysis. The expression of 480 genes differed by 2-fold or more between the two p53-infected cell lines. A number of potential targets for p53 were identified that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation. Proline oxidase, a mitochondrial enzyme involved in the proline/pyrroline-5-carboxylate redox cycle, was up-regulated by p53 in ECV but not in DECV cells. Pyrroline-5-carboxylate (P5C), a proline-derived metabolite generated by proline oxidase, inhibited the proliferation and survival of ECV-304 and DECV cells and induced apoptosis in both cell lines. A recombinant proline oxidase protein tagged with a green fluorescent protein at the amino terminus localized to mitochondria and induced apoptosis in p53-null H1299 non-small cell lung carcinoma cells. The results directly implicate proline oxidase and the proline/P5C pathway in p53-induced growth suppression and apoptosis.

A CBF5 mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing

In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.

Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes

Isopentenyl diphosphate (IPP) is the central intermediate in the biosynthesis of isoprenoids, the most ancient and diverse class of natural products. Two distinct routes of IPP biosynthesis occur in nature: the mevalonate pathway and the recently discovered deoxyxylulose 5-phosphate (DXP) pathway. The evolutionary history of the enzymes involved in both routes and the phylogenetic distribution of their genes across genomes suggest that the mevalonate pathway is germane to archaebacteria, that the DXP pathway is germane to eubacteria, and that eukaryotes have inherited their genes for IPP biosynthesis from prokaryotes. The occurrence of genes specific to the DXP pathway is restricted to plastid-bearing eukaryotes, indicating that these genes were acquired from the cyanobacterial ancestor of plastids. However, the individual phylogenies of these genes, with only one exception, do not provide evidence for a specific affinity between the plant genes and their cyanobacterial homologues. The results suggest that lateral gene transfer between eubacteria subsequent to the origin of plastids has played a major role in the evolution of this pathway.

Induction of tolerance to immunogenic tumor antigens associated with lymphomagenesis in HOX11 transgenic mice

Transgenic mice expressing human HOX11 in B lymphocytes die prematurely from lymphomas that initiate in the spleen and frequently disseminate to distant sites. Preneoplastic hematopoiesis in these mice is unperturbed. We now report that expression of the HOX11 transgene does not affect the ability of dendritic cells (DCs) to process and present foreign peptides and activate antigen-specific T cell responses. We also show that nontransgenic DCs presenting peptides derived from the human HOX11 protein are highly efficient stimulators of autologous T cells, whereas transgenic T cells are nonresponsive to peptides derived from the HOX11 transgene and the murine Meis1 protein. HOX11 transgenic mice thus show normal development of tolerance to immunogenic antigens expressed throughout B cell maturation. DCs pulsed with cell lysates prepared from lymphomas, obtained from HOX11 transgenic mice with terminal lymphoma, activate T cells from nontransgenic and premalignant transgenic mice, whereas T cells isolated from lymphomatous transgenic mice are nonresponsive to autologous tumor cell antigens. These data indicate that HOX11 lymphoma cells express tumor-rejection antigens that are recognized as foreign in healthy transgenic mice and that lymphomagenesis is associated with the induction of anergy to tumor antigen-specific T cells. These findings are highly relevant for the development of immunotherapeutic protocols for the treatment of lymphoma.

A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer–dimer interface of the tetrameric oligomerization domain of p53 (residues 325–355). Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state (tetramer, dimer, or equilibrium mixture of both) of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met (tetramer) > Ser Asp, His, Gln, > Glu, Lys (dimer), whereas the experimental results showed the following order: Met (tetramer) > Ser > Gln > His, Lys > Asp, Glu (dimers). For residue 344, the calculated trend was Leu (tetramer) > Ala > Arg, Gln, Lys (dimer), and the experimental trend was Leu (tetramer) > Ala, Arg, Gln, Lys (dimer). The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the α-helices at the dimer–dimer interface. Overall, this initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.

B7–CTLA4 interaction enhances both production of antitumor cytotoxic T lymphocytes and resistance to tumor challenge

Expression of B7-family costimulatory molecules CD80 (B7–1) and CD86 (B7–2) on tumor cells enhances host immunity. However, the role of the two B7 receptors, CD28 and CTLA4 (CD152), on T cells in antitumor immune response has not been clearly elucidated. Based on the effects of anti-CD28 and anti-CTLA4 mAbs on T cell response, it was proposed that CD28-B7 interaction promotes antitumor immunity, whereas B7-CTLA4 interaction down-regulates it. A critical test for the hypothesis is whether selective engagement of CTLA4 receptors by their natural ligands CD80 and CD86 enhances or reduces antitumor immunity. Here we used tumors expressing wild-type and mutant CD80, as well as mice with targeted mutation of CD28, to address this issue. We report that in syngeneic wild-type mice, B7W (W88>A), a CD80 mutant that has lost binding to CD28 but retained binding to CTLA4, can enhance the induction of antitumor cytotoxic T lymphocytes (CTL); B7Y (Y201>A), which binds neither CD28 nor CTLA4, fails to do so. Consistent with these observations, B7W-transfected J558 plasmocytoma and EL4 thymoma grow significantly more slowly than those transfected with either vector alone or with B7Y. Optimal tumor rejection requires wild-type CD80. Moreover, expression of a high level of CD80 on thymoma EL4 cells conveys immunity in mice with a targeted mutation of CD28 gene. Taken together, our results demonstrate that B7-CTLA4 interaction enhances production of antitumor CTL and resistance to tumor challenge and that optimal enhancement of antitumor immunity by CD80 requires its engagement of both CD28 and CTLA4.

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate–early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αVβ3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an αVβ3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

The MITE family Heartbreaker (Hbr): Molecular markers in maize

Transposable elements are ubiquitous in plant genomes, where they frequently comprise the majority of genomic DNA. The maize genome, which is believed to be structurally representative of large plant genomes, contains single genes or small gene islands interspersed with much longer blocks of retrotransposons. Given this organization, it would be desirable to identify molecular markers preferentially located in genic regions. In this report, the features of a newly described family of miniature inverted repeat transposable elements (MITEs) (called Heartbreaker), including high copy number and polymorphism, stability, and preference for genic regions, have been exploited in the development of a class of molecular markers for maize. To this end, a modification of the AFLP procedure called transposon display was used to generate and display hundreds of genomic fragments anchored in Hbr elements. An average of 52 markers were amplified for each primer combination tested. In all, 213 polymorphic fragments were reliably scored and mapped in 100 recombinant inbred lines derived from a cross between the maize inbreds B73 × Mo17. In this mapping population, Hbr markers are distributed evenly across the 10 maize chromosomes. This procedure should be of general use in the development of markers for other MITE families in maize and in other plant and animal species where MITEs have been identified.

Angiostatin gene transfer: Inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest

The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In in vitro studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G2/M transition induced by M-phase-promoting factors. AdK3-infected endothelial cells showed a marked mitosis arrest that correlated with the down-regulation of the M-phase phosphoproteins. A single intratumoral injection of AdK3 into preestablished rat C6 glioma or human MDA-MB-231 breast carcinoma grown in athymic mice was followed by a significant arrest of tumor growth, which was associated with a suppression of neovascularization within and at the vicinity of the tumors. AdK3 therapy also induced a 10-fold increase in apoptotic tumor cells as compared with a control adenovirus. Furthermore, we showed that systemic injection of AdK3 delayed C6 tumor establishment and growth, confirming that angiostatin can function in a paracrin manner. Our data support the concept that targeted antiangiogenesis, using adenovirus-mediated gene transfer, represents a promising alternative strategy for delivering antiangiogenic factors as their bolus injections present unsolved pharmacological problems.

Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: Role of vascular endothelial growth factor

The sequence of events that leads to tumor vessel regression and the functional characteristics of these vessels during hormone–ablation therapy are not known. This is because of the lack of an appropriate animal model and monitoring technology. By using in vivo microscopy and in situ molecular analysis of the androgen-dependent Shionogi carcinoma grown in severe combined immunodeficient mice, we show that castration of these mice leads to tumor regression and a concomitant decrease in vascular endothelial growth factor (VEGF) expression. Androgen withdrawal is known to induce apoptosis in Shionogi tumor cells. Surprisingly, tumor endothelial cells begin to undergo apoptosis before neoplastic cells, and rarefaction of tumor vessels precedes the decrease in tumor size. The regressing vessels begin to exhibit normal phenotype, i.e., lower diameter, tortuosity, vascular permeability, and leukocyte adhesion. Two weeks after castration, a second wave of angiogenesis and tumor growth begins with a concomitant increase in VEGF expression. Because human tumors often relapse following hormone–ablation therapy, our data suggest that these patients may benefit from combined anti-VEGF therapy.

Cytologically normal cells from neoplastic cervical samples display extensive structural abnormalities on IR spectroscopy: Implications for tumor biology

Fourier-transform IR (FT-IR) spectra of pelleted exfoliated cervical cells from patients with cervical cancer or dysplasia differ from those from normal women. To study the origin of these spectral changes, we obtained the FT-IR spectra of individual cervical cells from normal, dysplastic, and malignant cervical samples. Ninety five percent of normal superficial and intermediate cells displayed two distinct spectral patterns designated A and B, and 5% displayed an intermediate pattern, suggesting extensive structural heterogeneity among these cells. Parabasal and endocervical cells showed pattern B spectra. The spectra of malignant, dysplastic, and other abnormal cells also were characterized. Analysis of FT-IR spectra of over 2,000 individual cells from 10 normal females, 7 females with dysplasia, and 5 females with squamous cell carcinoma revealed that the spectra of normal-appearing intermediate and superficial cells of the cervix from women with either dysplasia or cancer differed from those of normal women. Chemometric and classical spectroscopic analysis showed a continuum of changes paralleling the transition from normalcy to malignancy. These findings suggest that (i) the structural changes underlying the spectroscopic changes are involved in or are a product of cervical carcinogenesis and (ii) the neoplastic process may be more extensive than currently recognized with morphological criteria. This approach may be useful for the structural study of neoplasia and also may be of help in the diagnosis or classification of cervical disorders.

Biosynthesis of the Pseudomonas polyketide coronafacic acid requires monofunctional and multifunctional polyketide synthase proteins

Coronafacic acid (CFA) is the polyketide component of the phytotoxin coronatine, a virulence factor of the plant pathogen Pseudomonas syringae. Our current knowledge of polyketide biosynthesis largely is based on the analysis of polyketide synthases (PKSs) in actinomycetes and other Gram-positive bacteria. Consequently, the cloning and characterization of the CFA biosynthetic gene cluster will contribute significantly to our knowledge of polyketide synthesis in Pseudomonas. In this report, we describe two genes in the CFA biosynthetic gene cluster that encode PKSs that are structurally and functionally similar to the multifunctional modular PKSs, which catalyze the synthesis of macrolide antibiotics. The CFA PKS genes were overproduced in Escherichia coli and shown to cross-react with antisera made to a modular PKS involved in erythromycin synthesis. A scheme for CFA biosynthesis is presented that incorporates the activities of all proteins in the CFA PKS. In this report a gene cluster encoding a pseudomonad polyketide has been completely sequenced and the deduced gene functions have been used to develop a biosynthetic scheme.

The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway

The PTEN/MMAC1 phosphatase is a tumor suppressor gene implicated in a wide range of human cancers. Here we provide biochemical and functional evidence that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive, dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1 expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its tumor suppressor function.

Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2Ek/Ek) was lethal in mid-gestation (the equivalent of mouse E9.5–E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long–Evans strain Tsc2Ek/Ek embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.

Desoxyepothilone B is curative against human tumor xenografts that are refractory to paclitaxel

The epothilones are naturally occurring, cytotoxic macrolides that function through a paclitaxel (Taxol)-like mechanism. Although structurally dissimilar, both classes of molecules lead to the arrest of cell division and eventual cell death by stabilizing cellular microtubule assemblies. The epothilones differ in their ability to retain activity against multidrug-resistant (MDR) cell lines and tumors where paclitaxel fails. In the current account, we focus on the relationship between epothilone and paclitaxel in the context of tumors with multiple drug resistance. The epothilone analogue Z-12,13-desoxyepothilone B (dEpoB) is >35,000-fold more potent than paclitaxel in inhibiting cell growth in the MDR DC-3F/ADX cell line. Various formulations, routes, and schedules of i.v. administration of dEpoB have been tested in nude mice. Slow infusion with a Cremophor-ethanol vehicle proved to be the most beneficial in increasing efficacy and decreasing toxicity. Although dEpoB performed similarly to paclitaxel in sensitive tumors xenografts (MX-1 human mammary and HT-29 colon tumor), its effects were clearly superior against MDR tumors. When dEpoB was administered to nude mice bearing our MDR human lymphoblastic T cell leukemia (CCRF-CEM/paclitaxel), dEpoB demonstrated a full curative effect. For human mammary adenocarcinoma MCF-7/Adr cells refractory to paclitaxel, dEpoB reduced the established tumors, markedly suppressed tumor growth, and surpassed other commonly used chemotherapy drugs such as adriamycin, vinblastine, and etoposide in beneficial effects.