Replacement of Fhit in cancer cells suppresses tumorigenicity

The candidate tumor suppressor gene, FHIT, encompasses the common human chromosomal fragile site at 3p14.2, the hereditary renal cancer translocation breakpoint, and cancer cell homozygous deletions. Fhit hydrolyzes dinucleotide 5′,5‴-P1,P3-triphosphate in vitro and mutation of a central histidine abolishes hydrolase activity. To study Fhit function, wild-type and mutant FHIT genes were transfected into cancer cell lines that lacked endogenous Fhit. No consistent effect of exogenous Fhit on growth in culture was observed, but Fhit and hydrolase “dead” Fhit mutant proteins suppressed tumorigenicity in nude mice, indicating that 5′,5‴-P1,P3-triphosphate hydrolysis is not required for tumor suppression.

Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type β signaling

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by endocrine tumors of parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene encodes a nuclear protein called menin. In MEN1 carriers inactivating mutations give rise to a truncated product consistent with menin acting as a tumor suppressor gene. However, the role of menin in tumorigenesis and its physiological functions are not known. Here, we show that menin inactivation by antisense RNA antagonizes transforming growth factor type β-mediated cell growth inhibition. Menin interacts with Smad3, and antisense menin suppresses transforming growth factor type β-induced and Smad3-induced transcriptional activity by inhibiting Smad3/4-DNA binding at specific transcriptional regulatory sites. These results implicate a mechanism of tumorigenesis by menin inactivation.

Increased histone H1 phosphorylation and relaxed chromatin structure in Rb-deficient fibroblasts.

Fibroblasts derived from embryos homozygous for a disruption of the retinoblastoma gene (Rb) exhibit a shorter G1 than their wild-type counterparts, apparently due to highly elevated levels of cyclin E protein and deregulated cyclin-dependent kinase 2 (CDK2) activity. Here we demonstrate that the Rb-/- fibroblasts display higher levels of phosphorylated H1 throughout G1 with the maximum being 10-fold higher than that of the Rb+/+ fibroblasts. This profile of intracellular H1 phosphorylation corresponds with deregulated CDK2 activity observed in in vitro assays, suggesting that CDK2 may be directly responsible for the in vivo phosphorylation of H1. H1 phosphorylation has been proposed to lead to a relaxation of chromatin structure due to a decreased affinity of this protein for chromatin after phosphorylation. In accord with this, chromatin from the Rb-/- cells is more susceptible to micrococcal nuclease digestion than that from Rb+/+ fibroblasts. Increased H1 phosphorylation and relaxed chromatin structure have also been observed in cells expressing several oncogenes, suggesting a common mechanism in oncogene and tumor suppressor gene function.

Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.

A maize cDNA encoding a member of the retinoblastoma protein family: involvement in endoreduplication.

Retinoblastoma (RB-1) is a tumor suppressor gene that encodes a 105-kDa nuclear phosphoprotein. To date, RB genes have been isolated only from metazoans. We have isolated a cDNA from maize endosperm whose predicted protein product (ZmRb) shows homology to the "pocket" A and B domains of the Rb protein family. We found ZmRb behaves as a pocket protein based on its ability to specifically interact with oncoproteins encoded by DNA tumor viruses (E7, T-Ag, E1A). ZmRb can interact in vitro and in vivo with the replication-associated protein, RepA, encoded by the wheat dwarf virus. The maize Rb-related protein undergoes changes in level and phosphorylation state concomitant with endoreduplication, and it is phosphorylated in vitro by an S-phase kinase from endoreduplicating endosperm cells. Together, our results suggest that ZmRb is a representative of the pocket protein family and may play a role in cell cycle progression. Moreover, certain plant monopartite geminiviruses may operate similarly to mammalian DNA viruses, by targeting and inactivating the retinoblastoma protein, which otherwise induces G1 arrest.

Apoptosis and APC in colorectal tumorigenesis.

Tumors result from disruptions in the homeostatic mechanisms that regulate cell birth and cell death. In colon cancer, one of the earliest manifestation of this imbalance is the formation of polyps, caused by somatic and inherited mutations of the adenomatous polyposis coli (APC) tumor suppressor gene in both humans and mice. While the importance of APC in tumorigenesis is well documented, how it functions to prevent tumors remains a mystery. Using a novel inducible expression system, we show that expression of APC in human colorectal cancer cells containing endogenous inactive APC alleles results in a substantial diminution of cell growth. Further evaluation demonstrated that this was due to the induction of cell death through apoptosis. These results suggest that apoptosis plays a role not only in advanced tumors but also at the very earliest stages of neoplasia.

Signature p53 mutation at DNA cross-linking sites in 8-methoxypsoralen and ultraviolet A (PUVA)-induced murine skin cancers.

A combination of psoralen and ultraviolet A radiation (PUVA) is widely used in the treatment of psoriasis. However, PUVA treatment increases the risk of developing skin cancer in psoriasis patients and induces skin cancer in mice. Since the DNA damage induced by PUVA is quite different from that induced by UV, we investigated whether PUVA-induced mouse skin cancers display carcinogen-specific mutations in the p53 tumor suppressor gene. The results indicated that 10 of 13 (77%) PUVA-induced skin tumors contained missense mutations predominantly at exons 6 and 7. In contrast, tumor-adjacent, PUVA-exposed skin from tumor-bearing animals did not exhibit p53 mutation in exons 4-8. Interestingly, about 40% of all mutations in PUVA-induced skin tumors occurred at 5'-TA sites, and an equal number of mutations occurred at one base flanking 5'TA or 5'-TAT sites. Since PUVA induces DNA cross-links exclusively at these sites and since UV "signature" mutations were rarely detected in PUVA-induced skin cancers, we can conclude that PUVA acts as a carcinogen by inducing unique PUVA signature mutations in p53. This finding may have implications for identifying the etiology of skin cancer in psoriasis patients who have undergone PUVA therapy.

The control of hematopoiesis and leukemia: from basic biology to the clinic.

Hematopoiesis gives rise to blood cells of different lineages throughout normal life. Abnormalities in this developmental program lead to blood cell diseases including leukemia. The establishment of a cell culture system for the clonal development of hematopoietic cells made it possible to discover proteins that regulate cell viability, multiplication and differentiation of different hematopoietic cell lineages, and the molecular basis of normal and abnormal blood cell development. These regulators include cytokines now called colony-stimulating factors (CSFs) and interleukins (ILs). There is a network of cytokine interactions, which has positive regulators such as CSFs and ILs and negative regulators such as transforming growth factor beta and tumor necrosis factor (TNF). This multigene cytokine network provides flexibility depending on which part of the network is activated and allows amplification of response to a particular stimulus. Malignancy can be suppressed in certain types of leukemic cells by inducing differentiation with cytokines that regulate normal hematopoiesis or with other compounds that use alternative differentiation pathways. This created the basis for the clinical use of differentiation therapy. The suppression of malignancy by inducing differentiation can bypass genetic abnormalities that give rise to malignancy. Different CSFs and ILs suppress programmed cell death (apoptosis) and induce cell multiplication and differentiation, and these processes of development are separately regulated. The same cytokines suppress apoptosis in normal and leukemic cells, including apoptosis induced by irradiation and cytotoxic cancer chemotherapeutic compounds. An excess of cytokines can increase leukemic cell resistance to cytotoxic therapy. The tumor suppressor gene wild-type p53 induces apoptosis that can also be suppressed by cytokines. The oncogene mutant p53 suppresses apoptosis. Hematopoietic cytokines such as granulocyte CSF are now used clinically to correct defects in hematopoiesis, including repair of chemotherapy-associated suppression of normal hematopoiesis in cancer patients, stimulation of normal granulocyte development in patients with infantile congenital agranulocytosis, and increase of hematopoietic precursors for blood cell transplantation. Treatments that decrease the level of apoptosis-suppressing cytokines and downregulate expression of mutant p53 and other apoptosis suppressing genes in cancer cells could improve cytotoxic cancer therapy. The basic studies on hematopoiesis and leukemia have thus provided new approaches to therapy.

Human semaphorins A(V) and IV reside in the 3p21.3 small cell lung cancer deletion region and demonstrate distinct expression patterns.

Semaphorins and collapsins make up a family of conserved genes that encode nerve growth cone guidance signals. We have identified two additional members of the human semaphorin family [human semaphorin A(V) and human semaphorin IV] in chromosome region 3p21.3, where several small cell lung cancer (SCLC) cell lines exhibit homozygous deletions indicative of a tumor suppressor gene. Human semaphorin A(V) has 86% amino acid homology with murine semaphorin A, whereas semaphorin IV is most closely related to murine semaphorin E, with 50% homology. These semaphorin genes are approximately 70 kb apart flanking two GTP-binding protein genes, GNAI-2 and GNAT-1. In contrast, other human semaphorin gene sequences (human semaphorin III and homologues of murine semaphorins B and C) are not located on chromosome 3. Human semaphorin A(V) is translated in vitro into a 90-kDa protein, which accumulates at the endoplasmic reticulum. The human semaphorin A(V) (3.4-kb mRNA) and IV (3.9- and 2.9-kb mRNAs) genes are expressed abundantly but differentially in a variety of human neural and nonneural tissues. Human semaphorin A(V) was expressed in only 1 out of 23 SCLCs and 7 out of 16 non-SCLCs, whereas semaphorin IV was expressed in 19 out of 23 SCLCs and 13 out of 16 non-SCLCs. Mutational analysis in semaphorin A(V) revealed mutations (germ line in one case) in 3 of 40 lung cancers. Our data suggest the need to determine the function of human semaphorins A(V) and IV in nonneural tissues and their role in the pathogenesis of lung cancer.

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in global DNA repair but exhibit normal transcription-coupled repair and enhanced UV resistance.

We investigated whether mutations in the p53 tumor suppressor gene alter UV sensitivity and/or repair of UV-induced DNA damage in primary human skin fibroblasts from patients with Li-Fraumeni syndrome, heterozygous for mutations in one allele of the p53 gene (p53 wt/mut) and sublines expressing only mutant p53 (p53 mut). The p53 mut cells were more resistant than the p53 wt/mut cells to UV cytotoxicity and exhibited less UV-induced apoptosis. DNA repair analysis revealed reduced removal of cyclobutane pyrimidine dimers from overall genomic DNA in vivo in p53 mut cells compared with p53 wt/mut or normal cells. However, p53 mut cells retained the ability to preferentially repair damage in the transcribed strands of expressed genes (transcription-coupled repair). These results suggest that loss of p53 function may lead to greater genomic instability by reducing the efficiency of DNA repair but that cellular resistance to DNA-damaging agents may be enhanced through elimination of apoptosis.

Growth suppression by p16ink4 requires functional retinoblastoma protein.

p16ink4 has been implicated as a tumor suppressor that is lost from a variety of human tumors and human cell lines. p16ink4 specifically binds and inhibits the cyclin-dependent kinases 4 and 6. In vitro, these kinases can phosphorylate the product of the retinoblastoma tumor suppressor gene. Thus, p16ink4 could exert its function as tumor suppressor through inhibition of phosphorylation and functional inactivation of the retinoblastoma protein. Here we show that overexpression of p16ink4 in certain cell types will lead to an arrest in the G1 phase of the cell cycle. In addition, we show that p16ink4 can only suppress the growth of human cells that contain functional pRB. Moreover, we have compared the effect of p16ink4 expression on embryo fibroblasts from wild-type and RB homozygous mutant mice. Wild-type embryo fibroblasts are inhibited by p16ink4, whereas the RB nullizygous fibroblasts are not. These data not only show that the presence of pRB is crucial for growth suppression by p16ink4 but also indicate that the pRB is the critical target acted upon by cyclin D-dependent kinases in the G1 phase of the cell cycle.

Loss of transformed phenotype in cancer cells by overexpression of the uteroglobin gene

Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2–13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (n = 25) remained apparently healthy even after 1½ years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.

In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis

We have developed a technique, methylation-specific PCR in situ hybridization (MSP-ISH), which allows for the methylation status of specific DNA sequences to be visualized in individual cells. We use MSP-ISH to monitor the timing and consequences of aberrant hypermethylation of the p16 tumor suppresser gene during the progression of cancers of the lung and cervix. Hypermethylation of p16 was localized only to the neoplastic cells in both in situ lesions and invasive cancers, and was associated with loss of p16 protein expression. MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D–Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D–Rb pathway during the evolution of an invasive cancer from precursor lesions. These studies demonstrate that MSP-ISH is a powerful approach for studying the dynamics of aberrant methylation of critical tumor suppressor genes during tumor evolution.

The BRCA2 gene product functionally interacts with p53 and RAD51

Germ-line mutations in the human BRCA2 gene confer susceptibility to breast cancer. Efforts to elucidate its function have revealed a putative transcriptional activation domain and in vitro interaction with the DNA repair protein RAD51. Other studies have indicated that RAD51 physically associates with the p53 tumor suppressor protein. Here we show that the BRCA2 gene product is a 460-kDa nuclear phosphoprotein, which forms in vivo complexes with both p53 and RAD51. Moreover, exogenous BRCA2 expression in cancer cells inhibits p53’s transcriptional activity, and RAD51 coexpression enhances BRCA2’s inhibitory effects. These findings demonstrate that BRCA2 physically and functionally interacts with two key components of cell cycle control and DNA repair pathways. Thus, BRCA2 likely participates with p53 and RAD51 in maintaining genome integrity.