Peripuberty stress leads to abnormal aggression, altered amygdala and orbitofrontal reactivity and increased prefrontal MAOA gene expression.

Although adverse early life experiences have been found to increase lifetime risk to develop violent behaviors, the neurobiological mechanisms underlying these long-term effects remain unclear. We present a novel animal model for pathological aggression induced by peripubertal exposure to stress with face, construct and predictive validity. We show that male rats submitted to fear-induction experiences during the peripubertal period exhibit high and sustained rates of increased aggression at adulthood, even against unthreatening individuals, and increased testosterone/corticosterone ratio. They also exhibit hyperactivity in the amygdala under both basal conditions (evaluated by 2-deoxy-glucose autoradiography) and after a resident-intruder (RI) test (evaluated by c-Fos immunohistochemistry), and hypoactivation of the medial orbitofrontal (MO) cortex after the social challenge. Alterations in the connectivity between the orbitofrontal cortex and the amygdala were linked to the aggressive phenotype. Increased and sustained expression levels of the monoamine oxidase A (MAOA) gene were found in the prefrontal cortex but not in the amygdala of peripubertally stressed animals. They were accompanied by increased activatory acetylation of histone H3, but not H4, at the promoter of the MAOA gene. Treatment with an MAOA inhibitor during adulthood reversed the peripuberty stress-induced antisocial behaviors. Beyond the characterization and validation of the model, we present novel data highlighting changes in the serotonergic system in the prefrontal cortex-and pointing at epigenetic control of the MAOA gene-in the establishment of the link between peripubertal stress and later pathological aggression. Our data emphasize the impact of biological factors triggered by peripubertal adverse experiences on the emergence of violent behaviors.

Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease.

Insertional mutagenesis represents a serious adverse effect of gene therapy with integrating vectors. However, although uncontrolled activation of growth-promoting genes in stem cells can predictably lead to oncological processes, this is far less likely if vector transcriptional activity can be restricted to fully differentiated cells. Diseases requiring phenotypic correction only in mature cells offer such an opportunity, provided that lineage/stage-restricted systems can be properly tailored. In this study, we followed this reasoning to design lentiviral vectors for the gene therapy of chronic granulomatous disease (CGD), an immune deficiency due a loss of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes, most often secondary to mutations in gp91(phox). Using self-inactivating HIV1-derived vectors as background, we first expressed enhanced green fluorescent protein (eGFP) from a minimal gp91(phox) promoter, adding various natural or synthetic transcriptional regulatory elements to foster both specificity and potency. The resulting vectors were assessed either by transplantation or by lentiviral transgenesis, searching for combinations conferring strong and specific expression into mature phagocytic cells. The most promising vector was modified to express gp91(phox) and used to treat CGD mice. High-level restoration of NADPH activity was documented in granulocytes from the treated animals. We propose that this lineage-specific lentiviral vector is a suitable candidate for the gene therapy of CGD.

Genomic instability in induced stem cells.

The ability to reprogram adult cells into stem cells has raised hopes for novel therapies for many human diseases. Typical stem cell reprogramming protocols involve expression of a small number of genes in differentiated somatic cells with the c-Myc and Klf4 proto-oncogenes typically included in this mix. We have previously shown that expression of oncogenes leads to DNA replication stress and genomic instability, explaining the high frequency of p53 mutations in human cancers. Consequently, we wondered whether stem cell reprogramming also leads to genomic instability. To test this hypothesis, we examined stem cells induced by a variety of protocols. The first protocol, developed specifically for this study, reprogrammed primary mouse mammary cells into mammary stem cells by expressing c-Myc. Two other previously established protocols reprogrammed mouse embryo fibroblasts into induced pluripotent stem cells by expressing either three genes, Oct4, Sox2 and Klf4, or four genes, OSK plus c-Myc. Comparative genomic hybridization analysis of stem cells derived by these protocols revealed the presence of genomic deletions and amplifications, whose signature was suggestive of oncogene-induced DNA replication stress. The genomic aberrations were to a significant degree dependent on c-Myc expression and their presence could explain why p53 inactivation facilitates stem cell reprogramming.

[The application of lentiviral vectors for tissue-specific gene manipulations].

The trophectoderm (TE) ofblastocysts, the first epithelium established in mammalian development, 1) plays signaling, supportive, and patterning functions during pre-implantation development, 2) ensures embryo implantation into the uterine wall, and 3) gives rise to extra-embryonic tissues essential for embryo patterning and growth after implantation. We show that mouse TE, itself permissive to lentiviral (LV) infection, represents a robust non-permeable physical barrier to the virus particles, thereby shielding the cells of the inner cell mass (ICM) from viral infection. This LV feature will allow modulations of gene expression in a lineage-specific manner, thus having significant applications in mouse functional genetics.

Adult rat liver cells transdifferentiated with lentiviral IPF1 vectors reverse diabetes in mice: an ex vivo gene therapy approach.

AIMS/HYPOTHESIS: We examined a clinical model of ex vivo transdifferentiation of primary adult hepatocytes to insulin-secreting cells for the treatment of type 1 diabetes. MATERIALS AND METHODS: Isolated rat hepatocytes were transduced in primary culture with a human lentivirus containing pancreatic duodenal homeobox 1 (PDX1, now known as insulin promoter factor 1, homeodomain transcription factor [IPF1]). Insulin expression and secretion of the newly engineered cells were assessed in vitro by RT-PCR, in situ hybridisation, immunostaining and radioimmunoassay. PDX1-transduced hepatocytes were further studied in vivo by injecting them under the renal capsule of diabetic SCID mice. RESULTS: Isolated rat hepatocytes were efficiently transduced with the lentiviral vector, as assessed by green fluorescent reporter gene expression. The transduced cells exhibited insulin at both mRNA (RT-PCR, in situ hybridisation) and protein levels (immunostaining and radioimmunoassay). Moreover, insulin secretion by the engineered cells was dependent on glucose and sulfonylurea. Other beta cell genes, including those encoding solute carrier family 2 (facilitated glucose transporter), member 2 (Slc2a2), glucokinase (Gck), ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (Abcc8), the potassium inwardly-rectifying channel, subfamily J, member 11 (Kcnj11) and proprotein convertase subtilisin/kexin type 1 (Pcsk1) were also expressed. The PDX1-transduced hepatocytes expressed several pancreatic transcription factors related to early pancreatic endocrine development (endogenous Pdx1, neurogenic differentiation factor 1 [Neurod1], and NK6 transcription factor related, locus 1 [Nkx6-1]) as well as the late-stage pancreatic transcription factors (paired box gene 4 [Pax4], paired box gene 6 [Pax6], and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A [Mafa]). Transplantation of 3 x 10(6) transdifferentiated liver cells under the renal capsule of seven streptozotocin-induced diabetic SCID mice resulted in significant reduction of non-fasting blood glucose levels from 30.7 +/- 1.3 to 8.7 +/- 3.7 mmol/l (mean +/- SEM, p = 0.01), in 6 to 8 weeks. Removal of the graft resulted in severe hyperglycaemia. CONCLUSIONS/INTERPRETATION: Ex vivo lentiviral-mediated PDX1 expression in isolated adult liver cells represents a potential model for type 1 diabetes mellitus therapy.

Treatment of fulminant liver failure by transplantation of microencapsulated primary or immortalized xenogeneic hepatocytes.

BACKGROUND: The aim of this study was to evaluate the in vitro and in vivo function of hepatocytes after immortalization, cryopreservation, encapsulation, and xenotransplantation into mice with fulminant liver failure (FLF). METHODS: Rat and human hepatocytes were isolated by collagenase digestion. Human hepatocytes were immortalized using lentiviral vectors. Rat and immortalized human hepatocytes (IHH) were encapsulated in 400 microm of alginate-poly-L-lysine (PLL; Sigma, Buchs, Switzerland)-alginate membranes and cryopreserved using a computerized device. In vitro, encapsulated hepatocytes (cryopreserved or noncryopreserved) were cultured; albumin secretion was measured by enzyme-linked immunosorbent assay. Microencapsulated (cryopreserved or noncryopreserved) hepatocytes were transplanted intraperitoneally to mice with FLF: group 1 (n = 10) transplantation of empty capsules; group 2 (n = 12) transplantation of free primary rat hepatocytes; group 3 (n = 12) transplantation of cryopreserved encapsulated rat hepatocytes; group 4 (n = 10) transplantation of encapsulated rat hepatocytes; group 5 (n = 9) transplantation of cryopreserved encapsulated IHH; group 6 (n = 10) transplantation of encapsulated IHH. RESULTS: Compared with free primary hepatocytes, cryopreserved or noncryopreserved encapsulated rodent hepatocytes showed similar levels of continuous in vitro albumin secretion over 1 week. Cryopreserved or noncryopreserved encapsulated IHH showed minimal albumin secretion compared with free primary human hepatocytes. Fulminant liver failure, produced by a combination of acetaminophen and 30% hepatectomy, resulted in a 20% to 30% host survival. In groups 1 and 2, survival was unmodified, compared with untreated mice. For groups 3 and 4, transplantation of cryopreserved or noncryopreserved encapsulated rat hepatocytes significantly increased survival rates to 66% and 80%, respectively (P < .01). For groups 5 and 6, transplantation of cryopreserved or noncryopreserved encapsulated IHH improved host survival to 50% and 55%, respectively (P < .05). CONCLUSIONS: Primary rodent hepatocytes maintained synthetic functions after encapsulation and cryopreservation. Immortalized human hepatocytes showed minimal albumin secretion in the absence of encapsulation and cryopreservation, suggesting that hepatocytes lose some specific functions after immortalization. After induction of FLF in mice, intraperitoneal transplantation of encapsulated (primary or immortalized, cryopreserved or noncryopreserved) xenogeneic hepatocytes significantly improved survival. These results indicate that naive and genetically modified hepatocytes can be successfully encapsulated, stored by cryopreservation, and transplanted into xenogeneic recipients with FLF to sustain liver metabolic functions.

Ectopic expression of the beta-cell specific transcription factor Pdx1 inhibits glucagon gene transcription.

AIMS/HYPOTHESIS: The transcription factor Pdx1 is required for the development and differentiation of all pancreatic cells. Beta-cell specific inactivation of Pdx1 in developing or adult mice leads to an increase in glucagon-expressing cells, suggesting that absence of Pdx1could favour glucagon gene expression by a default mechanism. METHOD: We investigated the inhibitory role of Pdx1 on glucagon gene expression in vitro. The glucagonoma cell line InR1G9 was transduced with a Pdx1-encoding lentiviral vector and insulin and glucagon mRNA levels were analysed by northern blot and real-time PCR. To understand the mechanism by which Pdx1 inhibits glucagon gene expression, we studied its effect on glucagon promoter activity in non-islet cells using transient transfections and gel-shift analysis. RESULTS: In glucagonoma cells transduced with a Pdx1-encoding lentiviral vector, insulin gene expression was induced while glucagon mRNA levels were reduced by 50 to 60%. In the heterologous cell line BHK-21, Pdx1 inhibited by 60 to 80% the activation of the alpha-cell specific element G1 conferred by Pax-6 and/or Cdx-2/3. Although Pdx1 could bind three AT-rich motifs within G1, two of which are binding sites for Pax-6 and Cdx-2/3, the affinity of Pdx1 for G1 was much lower as compared to Pax-6. In addition, Pdx1 inhibited Pax-6 mediated activation through G3, to which Pdx1 was unable to bind. Moreover, a mutation impairing DNA binding of Pdx1 had no effect on its inhibition on Cdx-2/3. Since Pdx1 interacts directly with Pax-6 and Cdx-2/3 forming heterodimers, we suggest that Pdx1 inhibits glucagon gene transcription through protein to protein interactions with Pax-6 and Cdx-2/3. CONCLUSION/INTERPRETATION: Cell-specific expression of the glucagon gene can only occur when Pdx1 expression extinguishes from the early alpha cell precursor.

Activity analysis of housekeeping promoters using self-inactivating lentiviral vector delivery into the mouse retina.

For most retinal degeneration disorders, no efficient treatment exists to preserve photoreceptors (PRs) and, consequently, to maintain vision. Gene transfer appears to be a promising approach to prevent PR loss. In order to design adequate vectors to target specific retinal cell types, we have analyzed the expression pattern of three different promoters (mouse phosphoglycerate kinase 1 (PGK), elongation factor-1 (EFS), rhodopsin (Rho)) in newborn and adult DBA/2 mice retinas using self-inactivating lentiviral vectors. At 7 days after intraocular injection and in optimal conditions, cell transduction was observed up to 1.5 mm from the injection site. PGK promoter expression was predominant in the retinal pigment epithelium (RPE), especially in adult mice, whereas the EFS promoter allowed a broad expression in the retina. Finally, as expected, the Rho promoter was specifically expressed in PRs. Differences in the cell types transduced and in transduction efficiency were observed between newborn and adult injected eyes emphasizing the importance of such basic studies for further gene therapy approaches as well as for understanding the transcriptional changes during retinal maturation. Thus, for future attempts to slow or rescue retinal degeneration by lentiviral delivery, PGK and EFS are more suitable to control the expression of a supporting secreted factor, PGK being mainly expressed in RPE and EFS in different cell types throughout the entire retina, whereas Rho should allow to specifically deliver the therapeutic gene to PRs.

Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vectors.

Lentiviral vectors represent an attractive technology platform from which to develop a targetable injectable gene delivery system for transduction of specific cell populations in vivo, irrespective of their cell cycle status. Targeted HIV-1-based lentiviral vectors were generated by pseudotyping them with chimeric murine leukemia virus (MLV) envelope glycoproteins displaying N-terminal targeting polypeptides. Vectors displaying an EGF polypeptide were fully infectious on EGF receptor-negative cells, but were inactive on cells with abundant EGF receptors (inverse targeting). Receptor-mediated inactivation of gene transfer was overcome by competing the EGF receptors on the target cells with soluble EGF or by removing the displayed EGF domain from the surface of the vector particles by factor Xa cleavage of a specific protease substrate engineered into its tethering linker (protease targeting). Intravenous infusion of nontargeted HIV-1 vectors led to maximal luciferase activity in liver and spleen with moderate or minimal activity in heart, skeletal muscle, lung, brain, kidney, ovaries and bone marrow. In contrast, intravenous EGF-displaying vectors were expressed maximally in spleen with very low level luciferase expression detectable in liver (EGF-receptor rich). Liver transduction by the EGF-displaying vector was restored by pretreating the animals with soluble EGF suggesting that these vectors are inversely targeted to spleen.

Inhibition of HIV-1 in cell culture by oligonucleotide-loaded nanoparticles.

PURPOSE: To investigate the potential use of polymeric nanoparticles for the delivery of antisense oligonucleotides in HIV-1-infected cell cultures. METHODS: Phosphorothioate oligonucleotides were encapsulated into poly (D,L-lactic acid) nanoparticles. Two models of infected cells were used to test the ability of nanoparticles to deliver them. HeLa P4-2 CD4+ cells, stably transfected with the beta-galactosidase reporter gene, were first used to evaluate the activity of the oligonucleotides on a single-round infection cycle. The acutely infected lymphoid CEM cells were then used to evaluate the inhibition of the viral production of HIV-1 by the oligonucleotides. RESULTS: The addition to infected CEM cells of nanoparticles containing gag antisense oligonucleotides in the nanomolar range led to strong inhibition of the viral production in a concentration-dependent manner. Similar results were previously observed in HeLa P4-2 CD4+ cells. Nanoparticle-entrapped random-order gag oligonucleotides had similar effects on reverse transcription. However, the reverse transcriptase activity of infected cells treated with nanomolar concentrations of free antisense and random oligonucleotides was not affected. CONCLUSIONS: These results suggest that poly (D,L-lactic acid) nanoparticles may have great potential as an efficient delivery system for oligonucleotides in HIV natural target cells, i.e., lymphocytic cells.

Transduction of CD34+ cells with lentiviral vectors enables the production of large quantities of transgene-expressing immature and mature dendritic cells.

BACKGROUND: Genetically engineered dendritic cells (DC) presenting specific antigens to T cells may be of great interest for immunotherapy. For this reason, the production of transgene-expressing DC derived from CD34 + cells transduced either shortly after ex vivo purification or during their differentiation into DC were evaluated. METHODS: CD34+ cells were transduced with lentivectors encoding for GFP before or after 21 days of culture with FLT3-ligand, thrombopoietin and stem cell factor and induction into DC with GM-CSF+IL-4 (G4) or G4+TNF (GT4). GFP and DC-specific marker expression was assessed by flow cytometry, and allostimulatory capacity was evaluated on GFP+ and GFP- sorted cells. RESULTS: Immature (G4-induced) DC obtained from amplified CD34 + cells were transducible by lentiviral vectors while mature (GT4-induced) DC were rather refractory. Moreover, since differentiated DC did not proliferate, large quantities of vectors were required to generate transgene-expressing cells with this protocol. In contrast, greater numbers of both immature and mature GFP- expressing DC were obtained with CD34+ cells exposed to lentivector shortly after purification. By the time of DC induction, GFP+ cells had increased by approximately 170-fold. After DC induction with G4, 32% of CD1a+, HLA-DR+, or CD40+ cells expressed GFP. CD1a+E-cadherin+ GFP+ Langerhans-like DC were also obtained. Incubation with TNF induced mature CD83+GFP+ DC that displayed a higher allostimulatory capacity than cells induced with G4 alone. CONCLUSION: The transduction of a small number of CD34+ cells with minimal doses of lentivector may allow for the production of a large number of DC expressing selected antigens useful for immunotherapy.

MNU induction of neoplasia in a platyfish model.

Interspecific hybrid crosses between members of the fish genus Xiphophorus have been used for over 70 years to study the genetic aspects of melanoma formation. In the well-established "Gordon-Kosswig" cross, the platyfish X. maculatus is outcrossed to the swordtail X. helleri, and the resulting backcross segregants spontaneously develop melanoma. We recently produced a distinct cross between X. maculatus and another platyfish species, X. couchianus. X. maculatus strain Jp 163 A is homozygous for several X-linked pigment pattern genes, including the Spotted dorsal (Sd), Dorsal red (Dr), and Anal fin spot (Af). Af is a sex-limited trait, coding exclusively for melanophores distributed on the modified anal fin or "gonopodium" in the adult male fish. Within F1 and BC1 hybrids (to X. couchianus), the Sd pigment pattern is phenotypically suppressed, whereas Dr and Af are enhanced. We exposed BC1 hybrids to the direct-acting carcinogen N-methyl-N-nitrosourea (MNU). Treatment led to the development of schwannomas, fibrosarcomas, and retinoblastomas. In addition, numerous MNU-treated males that inherited Af developed a pronounced melanotic phenotype, with melanin-containing cells oftentimes totally covering the gonopodium and extending further to grow within the ventral regions of the fish. Genetic linkage analysis of the BC1 hybrids revealed a significant (p < 0.01) association between CDKN2X genotype and the phenotypic degree of melanization. Such an association is consistent with a locus within linkage group V playing a role in the development of melanosis and delineates three genetic preconditions and a carcinogenic scheme resulting in melanosis of the ventral regions of hybrid fish. The overall study further alludes to the potential of using Xiphophorus fish to study carcinogenic mechanisms for tumors other than melanoma (schwannoma, fibrosarcoma, and retinoblastoma) and should enable extensive pathologic and molecular genetic studies of derived neoplastic abnormalities.

Cytoplasmic recruitment of INI1 and PML on incoming HIV preintegration complexes: interference with early steps of viral replication.

During the early phase of the retroviral life cycle, only a fraction of internalized virions end up integrating their genome into the chromosome, even though the resulting proviruses are almost systematically expressed. Here, we reveal that incoming retroviral preintegration complexes trigger the exportin-mediated cytoplasmic export of the SWI/SNF component INI1 and of the nuclear body constituent PML. We further show that the HIV genome associates with these proteins before nuclear migration. In the presence of arsenic, PML is sequestered in the nucleus, and the efficiency of HIV-mediated transduction is markedly increased. These results unveil a so far unsuspected cellular response that interferes with the early steps of HIV replication.

Living in oblivion: HIV immune evasion.

The human and simian immunodeficiency viruses (HIV and SIV, respectively) are members of the lentiviridae subgroup of retroviruses that cause a progressive failure of the host immunological functions culminating in the clinical collapse known as AIDS, or acquired immunodeficiency syndrome. In the absence of antiviral therapy, this course is inexorable in spite of an initially vigorous immune response. Two fundamental characteristics of the biology of primate lentiviruses explain this apparent paradox. First, HIV and SIV infect CD4(+)targets such as helper T lymphocytes and macrophages, that is, cells that normally play an essential role in the emergence and maintenance of an effective antiviral response. Second, these viruses have evolved a number of strategies to evade control by the immune system. These include mutational escape, latency, masking of antibody-binding sites on the viral envelope, downmodulation of the class I major histocompatibility complex (MHC-I), and upregulation of the Fas ligand on the surface of infected cells. Examining the mechanisms of these phenomena not only helps to understand how HIV wins its war against the immune system, but it also suggests as yet unexploited avenues to combat the virus through therapies and to develop a vaccine.

Lentiviral-mediated gene transfer into haematopoietic stem cells.

OBJECTIVES: Lentiviral vectors can transduce nondividing cells. As most haematopoietic stem cells (HSCs) are nondividing in vivo, lentiviral vectors are promising viral vectors to transfer genes into HSCs. DESIGN AND SETTING: We have used HIV-1 based lentiviral vectors containing the green fluorescent protein (GFP) gene to transduce umbilical cord blood CD34+ and CD34+/CD38- cells prior to transplantation into NOD/SCID mice. RESULTS: High level engraftment of human cells was obtained and transgene expression was seen in both myeloid and lymphoid lineages. Bone marrow from the primary transplant recipients mice was transplanted into secondary recipients. GFP expression was seen in both lymphoid and myeloid cells in the secondary recipients 6 weeks posttransplantation. Human haematopoietic progenitor colonies were grown from both primary and secondary recipients. Over 50% of the haematopoietic colonies in these recipients were positive for the GFP transgene by PCR. Following inverse PCR, amplified fragments were sequenced and integration of the vector into human genomic DNA was demonstrated. Several vectors containing different internal promoters were tested in NOD/SCID mice that had been transplanted with transduced CD34+ and CD34+/CD38- cells. The elongation factor-1alpha (EF-1alpha) promoter gave the highest level of expression, both in the myeloid and lymphoid progeny of the engrafting cells. CONCLUSIONS: These data collectively indicate that candidate human HSCs can be efficiently transduced with lentiviral vectors and that the transgene is highly expressed in their progeny cells.

Alternative oxidase in durum wheat mitochondria. Activation by pyruvate, hydroxypyruvate and glyoxylate and physiological role.

In order to gain a first insight into the alternative oxidase (AO) function in durum wheat mitochondria (DWM), we investigated some activation pathways of this enzyme in DWM purified from both etiolated shoots and green leaves. AO was activated when DWM were added with either pyruvate, known as an AO activator in other plant mitochondria, or alanine plus 2-oxoglutarate, which can generate intramitochondrial pyruvate and glutamate via transamination. In contrast, no AO activity was observed during oxidation of malate plus glutamate or succinate (which can generate malate). In this regard DWM differ from other plant mitochondria. Moreover, DWM were found: (i) to have a very low malic enzyme (ME) activity, (ii) to release oxaloacetate rather than pyruvate during malate oxidation and (iii) to poorly oxidise malate in the absence of glutamate, which removes oxaloacetate via transamination. Therefore, we show that, unlike other plant mitochondria, no pyruvate is generated inside DWM from malate via ME, allowing no AO activity. Other AO activators, alternative to pyruvate, were checked by evaluating the capability of several compounds to induce oxygen uptake and/or electrical membrane potential (Delta Psi) in cyanide-treated DWM. Hydroxypyruvate and glyoxylate, photorespiratory cycle intermediates, were found to be powerful AO activators, capable of inducing a maximal rate of cyanide-insensitive oxygen uptake 1.7 times and 2.3 times higher than pyruvate, respectively. These results suggest that in durum wheat a link may exist between AO activity and photorespiratory metabolism rather than malate metabolism. Moreover, we observed that AO activation resulted in both a partially coupled respiration and a reduction by half of the rate of superoxide anion generation; therefore, AO is expected to work as an antioxidative defence system when the photorespiratory cycle is highly active, as under environmental stress.

Genetic analysis of susceptibility to spontaneous and UV-induced carcinogenesis in Xiphophorus hybrid fish.

Xiphophorus interspecies hybrids provide genetically controlled models of tumor formation. Spontaneous melanomas form in first-generation backcross (BC(1)) hybrids produced from backcrossing F(1) hybrids derived from the platyfish X. maculatus Jp 163 A and the swordtail X. helleri to the X. helleri parental strain (the Gordon-Kosswig hybrid cross). Nodular melanomas originate in the dorsal fin from cells constituting the spotted dorsal (Sd) pigment pattern. A parallel genetic cross, with X. maculatus Jp 163 B, exhibits the spotted side (Sp) pigment pattern instead of Sd, and produces BC(1) hybrids exhibiting a much lower frequency of spontaneous melanoma formation. These hybrids are susceptible to melanoma development if irradiated with UV light as fry. Other hybrids involving these two strains of X. maculatus and different swordtail and platyfish backcross parents also have been investigated as potential tumor models, and show differing susceptibilities to UV-induced and spontaneous melanomas. Genotyping of individual BC(1) hybrids from several Xiphophorus crosses has implicated a locus, CDKN2X (a Xiphophorus homologue of the mammalian CDKN2 gene family, residing on Xiphophorus linkage group V), in enhancing pigmentation and the susceptibility to spontaneous and UV-induced melanoma formation in BC(1) hybrids from some crosses, but not others. Homozygosity for X. helleri and X. couchianus CDKN2X alleles in BC(1) hybrids can predispose individuals to melanoma, but this susceptibility is modified in other crosses depending both on the contributing sex-linked pigment pattern locus from X. maculatus (Sd or Sp), and the genetic constitution of the backcross parent. Xiphophorus BC(1) hybrids constitute unique genetic models offering the potential to analyze the contributions of specific genes to spontaneous and induced tumor formation in different, but comparable genetic backgrounds.

Genetic analysis of neoplasia induced by N-nitroso-N-methylurea in Xiphophorus hybrid fish.

Interspecific crosses within the genus Xiphophorus have historically been used to study the genetic aspects of melanoma formation. Melanomas typically occur as a result of deregulation of polymorphic, naturally occurring macromelanophore pigment patterns. Hybrid crosses also have been used to study the inducibility of melanoma by physical sources (such as UV light) and chemicals (such as N-methyl-N-nitrosourea, MNU). We previously defined a genomic region that is implicated in fish melanomagenesis and identified a candidate tumor suppressor gene (CDKN2X) within this genomic area. Highly significant associations between BC(1)-hybrid CDKN2X genotypes and UV-induced melanoma formation exist in a backcross produced from 2 inbred parental lines. However, when BC(1) hybrids are exposed to MNU as the tumor induction agent, a significant association between inheritance of CDKN2X alleles and tumor development is not observed. These data suggest there is mechanistic and genetic heterogeneity in melanomas derived from different etiologies within BC(1) hybrid fish.