Endothelial progenitor cells for cancer gene therapy.

Endothelial progenitor cells (EPCs) are promising for cancer therapy because they specifically target tumors. They have the capacity to home to, invade, migrate within and incorporate into tumor structures. They are easily expanded and can be armed with therapeutic payloads protected within the progenitor cells. Once in the tumor, armed EPCs can be triggered to induce cell death in surrounding tumor cells while being transiently protected from premature demise. In preclinical studies, therapeutic EPCs attenuated tumor growth and increased survival. Enhancing homing, self-protection and collateral tumor cell damage will increase the efficacy of EPCs for cancer gene therapy.

Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides.

An enhanced appreciation of uptake mechanisms and intracellular trafficking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeutic purposes. We addressed these issues by identifying cell surface proteins with which P-ODN specifically interact, studying P-ODN internalization mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques. Chemical cross-linking studies with a biotin-labeled P-ODN (bP-ODN), revealed the existence of five major cell surface P-ODN binding protein groups ranging in size from approximately 20-143 kD. Binding to these proteins was competitively inhibited with unlabeled P-ODN, but not free biotin, suggesting specificity of the interactions. Additional experiments suggested that binding proteins likely exist as single chain structures, and that carbohydrate moieties may play a role in P-ODN binding. Uptake studies with 35S-labeled P-ODN revealed that endocytosis, mediated by a receptor-like mechanism, predominated at P-ODN concentrations < 1 microM, whereas fluid-phase endocytosis prevailed at higher concentrations. Cell fractionation and ultrastructural analysis demonstrated the presence of ODN in clathrin coated pits, and in vesicular structures consistent with endosomes and lysosomes. Labeled ODN were also found in significant amounts in the nucleus, while none was associated with ribosomes, or ribosomes associated with rough endoplasmic reticulum (ER). Since nuclear uptake was not blocked by wheat germ agglutinin or concanavalin A, a nucleoporin independent, perhaps diffusion driven, import process is suggested. These data imply that antisense DNA may exert their effect in the nucleus. They also suggest rational ways to design ODN which might increase their efficiency.

Enriching suicide gene bearing tumor cells for an increased bystander effect.

The success of cancer gene therapies requiring in vivo gene transfer is severely hampered by the low efficacy of gene transfer, which has been difficult to improve. We therefore established a novel strategy to increase the share of transduced cells post gene transfer. We hypothesized that in vivo selection of tumor cells transduced with a suicide gene effectively enriches these cells within a tumor, thus allowing for an increased bystander effect after the prodrug is given, leading to enhanced eradication of tumor cells. We reasoned that in vivo enrichment should be achieved by exploiting the metabolism of the suicide gene product. For this 'enrichment-eradication' strategy we chose a fusion gene of cytosine deaminase and uracil phosphoribosyl transferase. Positive selection (enrichment) was to be achieved by concurrently giving N-(phosphonacetyl)-L-aspartate, an inhibitor of pyrimidine de novo synthesis, which leads to pyrimidine depletion-mediated death of non-transduced cells, and cytosine, to rescue fusion gene expressing cells via the pyrimidine salvage pathway. Negative selection (eradication) was to be induced by giving the prodrug 5-fluorocytosine. Indeed, murine NXS2 neuroblastoma cells transduced with the fusion gene were effectively enriched in vitro, leading to a near-complete bystander effect. In vivo enrichment-eradication of NXS2 cells led to decreased tumor growth. This proof-of-principle study shows that enrichment-eradication may compensate the effects of low in vivo gene transfer efficacy, a major obstacle in cancer gene therapy.

Cytosine deaminase/5-fluorocytosine gene therapy and Apo2L/TRAIL cooperate to kill TRAIL-resistant tumor cells.

The death ligand Apo2L/TRAIL (Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand) eradicates many tumor types while sparing most normal tissues. However, some tumors are resistant to TRAIL. We therefore determined if TRAIL cooperates with cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and investigated the mechanisms involved. Transfection of human LAN-5 neuroblastoma cells with CD rendered the cells (LAN-5-CD) sensitive to 5-FC-induced, caspase-dependent apoptosis. Mediated by caspase-3, CD/5-FC and TRAIL cooperated to induce apoptosis in these TRAIL-resistant cells and to cleave X-linked inhibitor of apoptosis protein (XIAP). In established LAN-5-CD tumors growing subcutaneously in mice, intratumorally applied TRAIL did not decrease tumor growth and systemically administered 5-FC only attenuated tumor growth. In contrast, 5-FC together with TRAIL dramatically decreased tumor growth and eradicated a tumor. Assuming sufficient gene transfer of CD in situ, CD/5-FC with TRAIL may be useful for the therapy of tumors resistant to TRAIL.

Mitochondrial amplification of death signals determines thymidine kinase/ganciclovir-triggered activation of apoptosis.

Previous clinical experience shows that the efficacy of suicide gene transfer in tumor therapy is limited, resulting from inefficient gene transfer or alternatively, from intrinsic resistance of the tumor in vivo. Herpes simplex virus thymidine kinase/ganciclovir (TK/GCV), a paradigmatic suicide gene therapy system, has been described to exert its cytotoxic effect, at least in part, by inducing apoptosis in target cells. Here, we report that mitochondria amplify TK/GCV-induced apoptosis by regulating p53 accumulation and the effector phase of apoptosis. Treatment with TK/GCV led to mitochondrial perturbations including loss of the mitochondrial membrane potential and release of cytochrome c from mitochondria into the cytosol, inducing caspase activation and nuclear fragmentation. Inhibition of TK/GCV-induced mitochondrial perturbations by Bcl-2 overexpression or by the mitochondrion-specific inhibitor bongkrekic acid also strongly inhibited TK/GCV-induced activation of caspases and apoptosis. TK/GCV-induced mitochondrial perturbations depended on caspase activity possibly initiated by death receptor signaling. Perturbation of mitochondrial function mediated accumulation of wild-type p53 protein, since Bcl-2 overexpression, bongkrekic acid, or inhibition of mitochondrial protein synthesis with chloramphenicol strongly reduced TK/GCV-induced accumulation of wild-type p53 protein. These findings suggest that TK/GCV therapy may be less efficient in tumors in which the mitochondrial amplification of TK/GCV-induced apoptosis is blocked, e.g., by Bcl-2 overexpression. Given the low efficacy of currently used gene therapy systems, our data on molecular mechanisms that regulate sensitivity or resistance toward TK/GCV-induced cytotoxicity might have important implications to improve the clinical application of suicide gene therapy.

No CDKN2 mutations in neuroblastomas.

Mutations of CDKN2 have been found recently in melanoma and many other tumor types. Neuroblastoma shares with melanoma a neuroectodermal origin and a high incidence of deletions of the short arm of chromosome 1. Therefore, we analyzed 18 primary neuroblastomas and 9 tumor-derived cell lines for mutations in CDKN2. We used PCR-single-strand conformation polymorphism to examine exons 1 and 2 of the CDKN2 gene for mutations, but none were detected. Furthermore, no homozygous deletions were detected and there was no loss of heterozygosity at the closely linked IFNA locus. We conclude that disruption of the CDKN2 gene is not required for malignant transformation of human neuroblastomas.

Significance of chromosome 1p loss of heterozygosity in neuroblastoma.

We analyzed 156 primary neuroblastoma tumor samples for loss of heterozygosity at the distal short arm of chromosome 1 (1p LOH). We also compared 1p LOH with known clinical and genetic prognostic variables as well as patient outcome. 1p LOH was detected in 30 of 156 tumors (19%) and was strongly associated with adverse clinical and biological features. 1p LOH was also strongly predictive of a poor outcome in univariate analyses (estimated 4-year survival, 32 +/- 10% SE versus 76 +/- 5% SE; P < 0.001). However, the prognostic value of 1p LOH was equivocal when stratified for amplification of the MYCN oncogene (P = 0.16). We conclude that 1p LOH is an important component of a pattern of genetic abnormalities in neuroblastoma associated with an aggressive clinical course.

Suicide gene therapy for pediatric tumors.

Tumor gene therapy is potentially very specific and efficacious. Suicide genes are promising tools in the arsenal of tumor gene therapy. However, problems of tumor targeting, low in vivo efficacy of nucleic acid transfer, and recent reports of adverse effects hinder the translation of this approach into clinical practice. Therefore vector design, tumor targeting, mechanisms of cell kill and killing of untransfected tumor cells must be improved. Once these problems are solved in vitro and in animal models, gene therapy holds great promise for pediatric oncology given the abundance of specific targets in pediatric tumors. This review describes the current state of preclinical research in tumor suicide gene therapy, provides an outline of pediatric suicide gene therapy protocols, and identifies potential targets in pediatric malignancies.

Human Krüppel-related 3 (HKR3): a candidate for the 1p36 neuroblastoma tumour suppressor gene?

Human Krüppel-related 3 (HKR3) is a zinc finger gene that maps within chromosome subbands 1p36.2-.3, a region postulated to contain a tumour suppressor gene associated with advanced neuroblastomas. Genomic clones of HKR3 were isolated from a P1 library and physically mapped to within 40 kb of D1S214 at 1p36.3. The gene is ubiquitously expressed in human tissues, but especially high levels are present in human fetal and adult nervous tissues. Hemizygous deletion of HKR3 in a lymphoblastoid cell line derived from a neuroblastoma patient with a constitutional 1p36 interstitial deletion and in the neuroblastoma cell line SK-N-AS, which also has a small interstitial 1p36 deletion, has been observed. Allelic loss at D1S214 in 15/15 informative primary neuroblastoma specimens with 1p36 deletions has also been observed. In a panel of 16 neuroblastoma cell lines, no gross genomic DNA rearrangements were noted, the gene was always expressed (albeit at variable levels) and there was no evidence for truncating mutations. Furthermore, there were no mutations detected in the zinc finger coding region in four neuroblastoma cell lines with 1p deletions analysed by direct sequence analysis. We conclude that HKR3 is a novel zinc finger gene that maps to a region of the genome commonly rearranged or deleted in neuroblastoma and other human cancers.

Molecular analysis of the region of distal 1p commonly deleted in neuroblastoma.

Cellular, cytogenetic, and molecular evidence indicates that chromosome band 1p36 is often deleted in neuroblastoma cell lines and tumours, suggesting the presence of one or more tumour suppressor genes in this region. We used a multifaceted approach to analyse the commonly deleted region, 28 distal 1p-specific polymorphic loci were used to detect loss of heterozygosity (LOH) in a panel of primary neuroblastoma tumours. Thirty-two of 122 tumours (26%) demonstrated LOH at three or more loci. In addition, a patient with a constitutional deletion of 1p36.2-.3 and two neuroblastoma cell lines with 1p36 abnormalities were characterised by FISH. When combined with the LOH data, a single consensus region of deletion was defined proximally by PLOD and distally by D1S80, a region spanning approximately five megabases. Several proposed candidate tumour suppressor genes, including ID3, CDC2L1, DAN, PAX7, E2F2, TNFR2 and TCEB3, map outside of this region; however, the transcription factor HKR3 cannot be excluded. LOH for 1p is correlated with adverse clinical and biological features and a poor prognosis, but 1p LOH is not an independent predictor of overall survival. To identify additional candidate genes, an integrated physical map of 1p35-36 is being constructed. The current map includes 445 polymerase chain reaction (PCR)-formatted markers and 608 YACs. This map will help identify region-specific transcripts by direct selection and sequencing.

N-myc augments death and attenuates protective effects of Bcl-2 in trophically stressed neuroblastoma cells.

N-myc has proapoptotic functions, yet it acts as an oncogene in neuroblastoma. Thus, antiapoptotic mechanisms have to be operative in neuroblastoma cells that antagonize the proapoptotic effects of N-myc. We conditionally activated N-myc in SH-EP neuroblastoma cells subjected to the trophic stress of serum or nutrient deprivation while changing the expression of Bcl-2, survivin and FLIP(L), antiapoptotic molecules often overexpressed in poor prognosis neuroblastomas. Bcl-2 protected SH-EP cells from death during nutritional deprivation by activating energetically advantageous oxidative phosphorylation. N-myc overrode the metabolic protection provided by Bcl-2-induced oxidative phosphorylation by reestablishing the glycolytic phenotype and attenuated the antiapoptotic effect of Bcl-2 during metabolic stress. Survivin partially antagonized the growth suppressive function of N-myc in SH-EP neuroblastoma cells during serum deprivation whereas FLIP(L) did not. These findings advance our understanding of the functions of N-myc in neuroblastoma cells.

Human blood late outgrowth endothelial cells for gene therapy of cancer: determinants of efficacy.

Human adult blood late outgrowth endothelial cells (BOECs) are potential yet untested cellular vehicles to target tumor-cytotoxic effectors to tumors. We show that, following intravenous injection into irradiated mice, BOECs home to Lewis lung carcinoma (LLC) lung metastases, but less so to liver or kidney metastases. BOECs targeted most but not all of the lung metastases, to a different degree. While most of the homed BOECs took up an extravascular position, some integrated into tumor vessels. Sequestration into normal tissue was low. Placental growth factor mediated both migration and invasion of BOECs into LLC spheroid masses in vitro, as did VEGF. When armed with a suicide gene, BOECs exerted a bystander effect on LLC cells in vitro and in vivo. Surprisingly, i.v. administration of armed BOECs into mice bearing multi-organ LLC metastases did not prolong survival. In addition to homing efficacy other parameters impacted upon the efficacy of BOECs. These include the ultimate susceptibility of BOECs to suicide gene-induced cell death, their paracrine proliferative effect on LLC cells and their low proliferation rate compared to LLC cells. Addressing these determinants may make BOECs a useful addition to the arsenal of tumor-targeting moieties.

[Tumour stem cells--a new concept in tumour biology]. / Tumorstammzellen--ein neues Konzept in der Tumorbiologie.

There is growing evidence that tumours display a hierarchy similar to normal tissues. Accordingly, a small population of tumour stem cells is supposed to perpetuate tumour growth. These cells renew themselves and are highly tumourigenic upon injection into immunocompromised animals, yielding tumours largely identical to those from which they were derived. Tumour stem cells exhibit a high degree of chemoresistance due to slow cycling and constitutive expression of multiple members of the ATP-BINDING CASSETTE (ABC) family of transporters. An increasing number of tumours with tumour stem cells has been identified by now, including breast, brain, colon and prostate cancers, as well as leukemias. Detailed characterization of tumour stem cells may contribute to a better understanding of the underlying biology of the respective tumours and lead to novel curative therapies.

Targeted release of oncolytic measles virus by blood outgrowth endothelial cells in situ inhibits orthotopic gliomas.

Malignant gliomas remain largely incurable despite intensive efforts to develop novel therapies. Replicating oncolytic viruses have shown great promise, among them attenuated measles viruses of the Edmonston B strain (MV-Edm). However, host immune response and the infiltrative nature of gliomas limit their efficacy. We show that human blood outgrowth endothelial cells (BOECs), readily expandable from peripheral blood, are easily infected by MV-Edm and allow replication of MV-Edm while surviving long enough after infection to serve as vehicles for MV-Edm (BOEC/MV-Edm). After intravenous and peritumoral injection, BOEC/MV-Edm deliver the viruses selectively to irradiated orthotopic U87 gliomas in mice. At the tumor, MV-Edm produced by the BOECs infect glioma cells. Subsequent spread from tumor cell to tumor cell leads to focal infection and cytopathic effects that decrease tumor size and, in the case of peritumoral injection, prolong survival of mice. Since MV-Edm within BOECs are not readily neutralized and because BOEC/MV-Edm search and destroy glioma cells, BOEC/MV-Edm constitute a promising novel approach for glioma therapy.

Sonography of subependymal cysts in congenital rubella syndrome.

Two newborns with congenital rubella syndrome are reported. Cranial sonography demonstrated bilateral cystic lesions in the subependymal germinal matrix. Congenital rubella and cytomegalovirus (CMV) infections are the most common proven causes of subependymal cysts of nonhaemorrhagic origin in the newborn. The sonographic detection of these cysts should prompt an intensive search for congenital viral infections.

[Effect of the place of birth on mortality and morbidity of the early form of group B streptococcal infection in newborn infants of normal weight]. / Einfluss des Geburtsortes auf Mortalität und Morbidität der Frühform der B-Streptokokkensepsis bei normalgewichtigen Neugeborenen.

In a retrospective analysis, 23 newborns (birth weight more than 2000 g) with early-onset group B streptococcal disease were studied. 13 babies had been born in another hospital and were subsequently transferred; these were compared with 10 infants from an adjacent perinatal centre. A paediatrician was present at the birth of the last group of infants, but only once at the birth of a baby born in a hospital not attached to our centre. At admission, the infants transferred from outside, were significantly more distressed, 5 infants died, whereas all infants of the second group survived. Although maternal risks or early signs of sepsis were present in 4 of the 5 decreased cases were in a state of septic shock at admission. To improve the neonatal outcome, the obstetrician must recognise the maternal risk factors, know the clinical picture of neonatal sepsis, and judge the symptoms accordingly. After basic diagnostic studies have been performed, intravenous penicillin must be administered and a qualified transfer to a neonatal intensive care unit must be arranged. Prevention of neonatal group B streptococcal disease is discussed.

Effectiveness of neonatal transport systems.

In order to assess the effectiveness of neonatal transport systems, morbidity on admission and early neonatal mortality of low birth weight infants below 2000 gm were studied. All infants referred to a neonatal department in Munich or Southern Bavaria from 1979 to 1981 were included. The data of infants born in Munich perinatal centers were compared to those of infants delivered in hospitals in the Munich area (radius 30 km) and in other hospitals in Southern Bavaria. Ninety-four percent of 248 LBW neonates born in the Munich perinatal centers, 87.5% of 736 infants and 84.4% of 681 LBW infants from the Munich area and Southern Bavaria respectively survived the first week of life although the morbidity risks of inborn infants were higher than those of the outborn. The presence of a pediatrician at birth and during neonatal transport to an NICU did not improve survival rates of infants delivered outside the perinatal centers. The effectiveness of neonatal transport systems is limited. They should be complemented by a maternal transport system, i.e., an infant transport in utero for cases in which the necessity for intensive neonatal care is expected.

A simple combined microdissection and aspiration device for the rapid procurement of single cells from clinical peripheral blood smears.

Molecular analysis of cells from cytology specimens can help to establish a diagnosis in ambiguous cases. However, mutations in heterogeneous samples might not be detected because of the diluting effect of DNA from normal background cells. Even if a mutation were detected, it could not be traced back to a specific cell type. Molecular analysis of single cells circumvents this problem. Both mechanical and laser assisted methods have been described for the selective procurement of cells from histology slides; however, they have the drawback of either being technically demanding or expensive. Furthermore, it is nuclear whether they can be applied to cytology specimens. Finally, few of these techniques are able to procure single cells. Therefore, we developed a simplified combined microdissection and aspiration device for the rapid procurement of single cells from clinical cytology specimens. The principle of this device, called the cytopicker, is the combination of the microdissection tool, a steel cannula, with the aspiration tool, a glass capillary connected to a vacuum, into one device. Steel cannulae are optimal for microdissection of cells from the hard matrix of cytology specimens but aspirate poorly. On the other hand, glass capillaries are suboptimal for dissecting but aspirate very well. Combining both tools into one by inserting the capillary into the cannula allows optimal dissection using the cannula (with the glass capillary with-drawn and thus protected), followed by optimal aspiration using the capillary (after being advanced through the cannula). All movements of the device are controlled by just one micromanipulator, making the cytopicker inexpensive to manufacture. The cytopicker can rapidly and simply procure single cells, such as lymphoblasts, from cytology specimens, such as peripheral blood smears. DNA from these cells can be amplified by PCR. However, precautions have to be taken to avoid contamination. Once improved further, the cytopicker might facilitate molecular analysis in the routine cytology laboratory.

Murine models for experimental therapy of pediatric solid tumors with poor prognosis.

Novel therapeutic strategies are required for pediatric solid tumors with poor prognosis such as metastasizing neuroblastoma, rhabdomyosarcoma and Ewing's sarcoma. A prerequisite for the development of such new therapies is the availability of murine models. To be useful for therapeutic studies, these models should not only recapitulate the genetic alterations characteristic of the human disease but should also mimic the metastatic process and the response to current therapy, both of which ultimately determine the fate of children with these tumors. This review scrutinizes the utility of existing murine models of neuroblastoma, rhabdomyosarcoma and Ewing's sarcoma for investigating novel therapies. Much experience has been gained with both syngeneic and xenogeneic transplantable models of these tumors, while transgenic and knockout mice are just beginning to be available for therapeutic investigations. Modeling the genetic aberrations characterizing these tumors may provide faithful models for therapeutic studies in the future.

Whole genome amplification of single cells from clinical peripheral blood smears.

Molecular analysis of clinical samples has been hampered by the lack of fresh or frozen specimens and the presence of contaminating background cells within samples obscuring the molecular analysis of the pathological cells of interest. Routine cytology specimens are a ubiquitous and abundant, yet largely untapped, source of clinical samples for molecular analysis. Morphologically defined single cells from peripheral blood smears can be microdissected from contaminating background cells and their whole genome amplified by primer extension preamplification, followed by polymerase chain reaction analysis of the specific DNA of interest. Thus, molecular information can be traced back to the cell of origin in these clinical specimens. This should allow studies on clonality, loss of heterozygosity, mutation, or amplification of multiple loci from one single cell in haematological smears and possibly other clinical cytology specimens.