Prospective evaluation of biweekly streptozotocin in 19 dogs with insulinoma.

BACKGROUND: Administration of streptozotocin (STZ) at a 21-day interval has been described in dogs with stage II and III insulinoma. Myelosuppression was not observed, suggesting the possibility of increasing dose intensity by decreasing the interval between doses. OBJECTIVE: To describe the tolerability of a biweekly STZ protocol. A secondary objective was to describe the outcome of dogs treated with this protocol. ANIMALS: Nineteen dogs with residual local, metastatic, or recurrent insulinoma. METHODS: After surgery for insulinoma, or at the time of recurrence, dogs were treated with a previously described STZ and saline diuresis protocol. Treatments were administered every 14 days. All dogs received antiemetic treatment. Adverse events (AEs) were recorded and graded. Outcome endpoints assessed were progression-free survival (PFS) and survival. RESULTS: None of the dogs experienced neutropenia or thrombocytopenia. Mild to moderate gastrointestinal toxicity was the most common AE. Diabetes mellitus was observed in 8 dogs and, in 6, resulted in euthanasia or death. Two dogs developed nephrotoxicity manifested as Fanconi syndrome in 1 and nephrogenic diabetes insipidus in the other. Six dogs developed increased alanine amino transferase activity. Hypoglycemia at the end of the STZ infusion, resulted in collapse in 1 dog and a generalized seizure in another. The median overall PFS and survival time were 196 and 308 days, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Streptozotocin can be safely administered to dogs with insulinoma, but serious AEs are possible. Additional investigation is required to better define the role of STZ in managing dogs with insulinoma.

An open-label, dose-escalating phase I study of elsamitrucin (SPI 28090) in treatment of malignant solid tumors in dogs.

BACKGROUND: Elsamitrucin, the most potent topoisomerase II inhibitor available, is unique in that it does not cause neutropenia or cardiotoxicosis. It has antitumor activity in human patients with relapsed or refractory non-Hodgkin's lymphoma. OBJECTIVES: To determine the maximum tolerated dose (MTD), safety, and toxicity of elsamitrucin when administered to tumor-bearing dogs and to evaluate the incidence and severity of adverse events. ANIMALS: Twenty client-owned dogs with spontaneous malignant solid tumors or lymphoma that were refractory to, or for which the owner declined, conventional therapy were enrolled. METHODS: Prospective, open-label, single-agent study. Escalating doses of elsamitrucin were administered once weekly i.v. for up to 16 weeks in a modified 3 + 3 Phase I design. The starting dose was 0.06 mg/kg with escalation to 0.08 and 0.09 mg/kg. Dogs that remained on the study were monitored for evidence of toxicoses for at least 4 weeks and for survival every 2 months. RESULTS: Serious adverse events (SAEs) possibly attributable to elsamitrucin include: 1 dog developed heart failure and another developed hepatotoxicosis manifested by increased alanine aminotransferase, alkaline phosphatase, and total bilirubin (0.06 mg/kg dose); 1 dog developed severe anorexia and diarrhea, another developed severe diarrhea alone, and a 3rd dog went into cardiac arrest (0.09 mg/kg dose). A dose of 0.08 mg/kg was well tolerated with no SAEs. CONCLUSIONS AND CLINICAL IMPORTANCE: The MTD and recommended dose for Phase II trials of elsamitrucin is 0.08 mg/kg i.v. weekly. Elsamitrucin might be considered for combination protocols with myelosuppressive chemotherapy agents.

Deregulated cyclin E promotes p53 loss of heterozygosity and tumorigenesis in the mouse mammary gland.

Deregulation of cyclin E expression and/or high levels have been reported in a variety of tumors and have been used as indicators of poor prognosis. Although the role that cyclin E plays in tumorigenesis remains unclear, there is evidence that it confers genomic instability when deregulated in cultured cells. Here we show that deregulated expression of a hyperstable allele of cyclin E in mice heterozygous for p53 synergistically increases mammary tumorigenesis more than that in mice carrying either of these markers individually. Most tumors and tumor-derived cell lines demonstrated loss of p53 heterozygosity. Furthermore, this tumor susceptibility is related to the number of times the transgene is induced indicating that it is directly attributable to the expression of the cyclin E transgene. An indirect assay indicates that loss of p53 function is an early event occurring in the mammary epithelia of midlactation mammary glands in which cyclin E is deregulated long before evidence of malignancy. These data support the hypothesis that deregulated expression of cyclin E stimulates p53 loss of heterozygosity by promoting genomic instability and provides specific evidence for this in vivo. Cyclin E deregulation and p53 loss are characteristics often observed in human breast carcinoma.

Genomic instability in Gadd45a-deficient mice.

Gadd45a-null mice generated by gene targeting exhibited several of the phenotypes characteristic of p53-deficient mice, including genomic instability, increased radiation carcinogenesis and a low frequency of exencephaly. Genomic instability was exemplified by aneuploidy, chromosome aberrations, gene amplification and centrosome amplification, and was accompanied by abnormalities in mitosis, cytokinesis and growth control. Unequal segregation of chromosomes due to multiple spindle poles during mitosis occurred in several Gadd45a -/- cell lineages and may contribute to the aneuploidy. Our results indicate that Gadd45a is one component of the p53 pathway that contributes to the maintenance of genomic stability.

Overproduction of MDM2 in vivo disrupts S phase independent of E2F1.

Expression of a beta-lactoglobulin (BLG)/mdm2 transgene (BLGmdm2) in the epithelial cells of the mouse mammary gland causes an uncoupling of S phase from M phase, resulting in polyploidy and tumor formation. The cell cycle defects are independent of interactions with p53. Because MDM2 also binds and activates the S phase-specific transcription factor E2F1, we hypothesized that increased E2F1 activity causes the development of the BLGmdm2 phenotype. We, therefore, generated BLGmdm2 mice that were null for E2F1. We observed no notable differences in histology or cyclin gene expression between BLGmdm2 and BLGmdm2/E2F1-/- mice, indicating that endogenous E2F1 activity was not required for the BLGmdm2 phenotype. Because, depending on the experimental system, either loss of E2F1 function or overexpression of E2F1 results in transformation, we also tested whether overexpression of E2F1 augmented the severity of the BLGmdm2 phenotype by generating mice that were bitransgenic for BLGmdm2 and BLGE2F1. We observed a unique mixture of the two single transgenic phenotypes histologically and found no significant changes in cyclin levels, indicating that overexpression of E2F1 had no effect on the BLGmdm2 transgenic phenotype. Thus, increased expression or absence of E2F1 does not affect the ability of MDM2 to disrupt the cell cycle.

Gene knockout and transgenic technologies in risk assessment: the next generation.

Transgenic and knockout mice have been proposed as substitutes for one of the standard 2-yr rodent assays. The advantages of using genetically engineered mouse models is that fewer mice are needed, the time to develop disease is greatly reduced, and the mice are predisposed to developing cancer by virtue of gain or loss of functions. The models currently being used have yielded a large amount of data and have proved to be informative for risk assessment; however, they are still far from ideal. In fact, they inherently do not reflect the complexity of mutation and carcinogenesis in humans. Recent advances in technology and the creation of new knockout mice may produce more useful and more sensitive models. This review covers two recent advances in technology--inducible and regulatable gene expression and targeted genetic modifications in the genome--that will allow us to make better models. I also discuss new gene deletion and transgenic mouse models and their potential impact on risk-assessment assays. These models are presented in the context of four basic components or events that occur in the multistep process leading to cancer: maintenance of gene expression patterns, genome stability and DNA repair, cell-cell communication and signaling, and cell-cycle regulation. Finally, surrogate markers and utility in risk assessment are also discussed. This review is meant to stimulate further discussion in the field and to generate excitement about working toward the next generation of risk-assessment models.

Targeted expression of MDM2 uncouples S phase from mitosis and inhibits mammary gland development independent of p53.

MDM2 is a cellular protein that binds to and inactivates the p53 tumor suppressor protein. Although mdm2 has been shown to function as an oncogene in vitro, all studies to date have assessed MDM2 activities in the presence of p53, implicating p53 inactivation in MDM2-directed transformation. To determine the role of MDM2 in the cell cycle and in tumorigenesis and whether or not this role is dependent on p53, an MDM2 minigene was expressed during gestation and lactation in the mammary gland of both wild-type p53 (p53+/+) and p53 knockout (p53-/-) mice using the bovine beta-lactoglobulin promoter. In six different transgenic mouse lines, deregulated expression of MDM2 inhibited normal development and morphogenesis of the mammary gland, and caused cellular hypertrophy and nuclear abnormalities. These abnormalities included both multinucleated cells and enlarged cells with giant nuclei. Although there were fewer epithelial cells present in the transgenic mammary gland, no apoptosis was observed. Instead, BrdU incorporation and PCNA staining showed that 12%-27% of the transgenic mammary epithelial cells were in S phase at a time when normal cells were terminally differentiated. Analysis of DNA content showed that 30%-45% of the cells were polyploid, with DNA contents up to 16N, indicating that overexpression of MDM2 caused mammary epithelial cells to undergo multiple rounds of S phase without cell division. This phenotype was similar in the p53+/+ and p53-/- background, demonstrating a role for MDM2 in the regulation of DNA synthesis that is independent of the ability of MDM2 to inhibit p53 activity. Additionally, multiple lines of BLGMDM2 transgenic mice developed mammary tumors, confirming that overproduction of MDM2 contributes to tumorigenesis in epithelial cells in vivo.

Inflammatory polyarthropathy and bone remodeling in HTLV-I Tax-transgenic mice.

Transgenic mice carrying the tax gene of human T-cell lymphotropic virus type I displayed a high prevalence of arthropathy. The percentage of affected animals increased with age, reaching a peak of 43% at 20 months. Southern analysis of deoxyribonucleic acid (DNA) from tissue samples indicated that disease development was related to tax copy number. Histopathologic evaluation of the ankle joints disclosed deep erosion of the synovial lining, fibrous tissue proliferation together with angiogenesis, mononuclear cell infiltration, and activation of osteoclasts. Radiologic examination confirmed joint involvement and revealed bone architecture modifications. The phenotype exhibited by the affected animals closely resembles that of seronegative arthritis in humans, and may indicate tax protein as a causal agent of arthropathy observed in HTLV-I infected individuals.

Activation of erbB2 and c-src in phorbol ester-treated mouse epidermis: possible role in mouse skin tumor promotion.

In recent work we showed that the EGF receptor (EGFr) was activated in tumor promoter treated mouse epidermis (Cell Growth & Differentiation, 6: 1447-1455, 1995). In the present study, we have investigated the possible role of other erbB family members in the process of tumor promotion. Both erbB2 and erbB3, but not erbB4, were expressed in cultured mouse keratinocytes and in mouse epidermis in vivo. In cultured mouse keratinocytes, EGF stimulated rapid tyrosine phosphorylation of erbB2 followed by a time-dependent degradation of erbB2 protein. Furthermore, an increase in erbB2:EGFr heterodimer formation was also induced by EGF. In contrast to the results with erbB2, EGF did not induce tyrosine phosphorylation, the degradation of erbB3, or erbB3:EGFr heterodimer formation in cultured keratinocytes. Further analyses revealed that c-src kinase activity was dramatically elevated in cultured mouse keratinocytes exposed to EGF. In mouse epidermis following multiple treatments with 12-O-tetradecanoylphorbol-13-acetate (TPA), the phosphotyrosine content of erbB2 was significantly elevated in a dose-dependent manner. Concomittantly, erbB2:EGFr heterodimer formation and c-src kinase activity were also elevated in TPA-treated epidermis. Structure-activity relationships with several phorbol ester analogs showed that the elevated phosphorylation of erbB2 in mouse epidermis followed closely with tumor promoting ability. Activation of erbB2 and c-src kinase were also observed in the epidermis of TGF alpha transgenic mice where expression of human TGF alpha was targeted to basal keratinocytes with the human K14 promoter. Collectively, the current data suggest that the activation of erbB2 in phorbol ester treated skin can be explained solely by a mechanism involving elevation of EGFr ligands and activation of the EGFr. In addition, activation of c-src may be an important downstream effector in mouse keratinocytes both in vivo and in vitro, following activation of the EGFr, erbB2, or both.

Induction of mammary gland hyperplasia and carcinomas in transgenic mice expressing human cyclin E.

Deregulated expression of several cell cycle regulatory genes has been demonstrated to be associated with cancer. In particular, a strong correlation has been established between inappropriate cyclin E expression and human breast cancer. To determine the ability of cyclin E to play a causative role in mammary tumorigenesis, regulatory sequences from the ovine beta-lactoglobulin gene were utilized to specifically target expression of human cyclin E to the mammary glands of pregnant and lactating mice. Lactating mammary glands of transgenic mice expressing cyclin E contained areas of hyperplasia, primarily papillary projections of hyperplastic cells, which were rarely observed in lactating glands of control mice. Over 10% of female cyclin E transgenic mice have developed mammary carcinomas, with latencies ranging from 8 to 13 months. Tumor analysis revealed the presence of transgene-specific cyclin E RNA and protein, as well as cyclin E- and cdk2-associated kinase activity, suggesting that cyclin E is likely a contributing component of tumorigenic progression in this model system.

Neurodegenerative changes including altered tau phosphorylation and neurofilament immunoreactivity in mice transgenic for the serine/threonine kinase Mos.

Transgenic mice expressing the oncogenic protein-serine/threonine kinase Mos at high levels in the brain display progressive neuronal degeneration and gliosis. Gliosis developed in parallel with the onset of postnatal transgene expression and led to a dramatic increase in the number of astrocytes positive for GFAP, vimentin, and possibly tau. Interestingly, vimentin is normally expressed only in immature or neoplastic astrocytes, but appears to be induced to high levels in Mos-transgenic, mature astrocytes. Mos can activate mitogen activated protein kinase (MAPK) and MAPK has been implicated in Alzheimer-type tau phosphorylation. In the Mos-transgenic brain we found increased levels of phosphorylation at one epitope on tau containing serines 199 and 202 (numbering according to human tau), a pattern similar but not identical to that found in Alzheimer's disease. In addition, Mos-transgenic mice express a novel neurofilament-related protein that might be a proteolytic neurofilament heavy chain degradation product. These results suggest that activation of protein phosphorylation in neurons can result in changes in cytoskeletal proteins that might contribute to neuronal degeneration.

Overexpression of an Agouti cDNA in the skin of transgenic mice recapitulates dominant coat color phenotypes of spontaneous mutants.

The classical mouse fancy Agouti gene is responsible for the wild-type coat color where hairs are banded black and yellow. The Agouti gene encodes a 131-amino-acid secreted protein product that regulates phaeomelanin synthesis by melanocytes in mice. Mice with a dominant mutation at this locus, Ay, develop a yellow coat color, obesity, and diabetes, as the result of a deletion that results in ectopic overexpression of the Agouti gene mRNA in all tissues examined. Obesity and diabetes in Ay mutant mice could be caused by circulation of the protein, or localized action in specific tissues as a paracrine factor acting in cell-cell communication. To test these two possibilities, the Agouti cDNA was overexpressed in the skin of transgenic mice using either the Tyrosinase-Related Protein-1 or the keratin-14 (K14) promoter, the latter with and without an intron. The K14 promoter directed high constitutive levels of expression of Agouti mRNA in the skin, and several lines of transgenic mice exhibited coat colors resembling dominant Agouti allele phenotypes. Two highly expressing K14-Agouti transgenic lines, with light-yellow pelage, were analyzed for obesity and hyperglycemia. The transgenic mice were not significantly different from the controls (P > 0.05), indicating that the Agouti product does not act as an endocrine factor. RNase protection assays revealed a correlation between the levels of dorsal and ventral skin expression with pigmentation/phaeomelanin phenotypes. Co-injection experiments with the Agouti transgenes and other transgenes demonstrated co-integration of the two constructs at the same chromosomal site in approximately 95% of F1 progeny, allowing transgene inheritance to be visibly detected.

Overexpression of cyclin A in the mammary glands of transgenic mice results in the induction of nuclear abnormalities and increased apoptosis.

Aberrant expression of several cyclin genes has been demonstrated to be associated with many types of tumors. To determine the capacity of cyclin A to function as an oncogene in vivo, wild-type and mutant cyclin A proteins were specifically overexpressed in the mammary glands of transgenic mice using regulatory sequences from the ovine beta-lactoglobulin gene. Several lines of transgenic mice were generated that expressed human cyclin A or a nondegradable mutant version of human cyclin A, in which the amino-terminal 89 amino acids encompassing the cyclin destruction box were removed. The cyclin A transgene products were localized in the nuclei of mammary epithelial cells, and the transgenic mammary glands had an increase in cyclin A- and cdk2-associated H1 kinase activity. Many mammary epithelial cells in the transgenic glands exhibited nuclear abnormalities, including multinucleation and karyomegaly, which were suggestive of preneoplastic alterations. The abnormalities were more severe in mammary glands of the mutant cyclin A transgenics, which expressed a stabilized cyclin A protein. In situ analysis of mid-lactation mammary gland sections revealed increased numbers of apoptotic cells in the transgenic glands. Double transgenic animals were generated that expressed both the mutant human cyclin A and human cdk2 transgenes, and a more pronounced phenotype resulted. The bigenic mammary glands exhibited focal areas of hyperplasia, as well as a greater incidence of apoptosis than observed in the single transgenic glands, demonstrating in vivo cooperation between these genes in transformation and apoptotic signaling pathways.

Expression of the v-Mos oncogene in male meiotic germ cells of transgenic mice results in metaphase arrest.

To explore the role of pp39mos in male germ cell meiosis, we have constructed transgenic mice carrying either the c-Mos or v-Mos genes linked to the human male germ cell-specific phosphoglycerate kinase-2 promoter. All male transgenic mice bearing the v-Mos but not the c-Mos construct were sterile due to arrest of germ cells at metaphase I. Immunocytochemistry performed on sections from control and c-Mos transgenic testes with eight different monoclonal and polyclonal antisera against either alpha-, beta- or gamma-tubulins demonstrated that all could recognize MI spermatocyte spindles from control and c-Mos transgenics, but only one monoclonal anti-microtubule sera decorated the spindles of v-Mos-arrested meiotic figures. Western blot analyses with this one serum revealed a change in proteins in the v-Mos samples. Immunocytochemistry with the MPM-2 monoclonal antibody, which is specific for epitopes phosphorylated during mitosis, demonstrated an increase in cytoplasmic and spindle-associated phosphoproteins in arrested v-Mos spermatocytes. Western analysis with MPM-2 showed an increase in a M(r) 50,000-55,000 and a M(r) 25,000-29,000 protein in Mos transgenic testes when compared to controls. An anti-MAP kinase antibody demonstrated an increase in all four MAP kinases in testes of transgenic mice. Thus, overexpression of pp39v-mos during male germ cell meiosis resulted in an alteration of various cell cycle related kinases and cytostatic factor-like arrest at MI.

Delayed rectifier K+ channel overexpression in transgenic islets and beta-cells associated with impaired glucose responsiveness.

Glucose stimulation of pancreatic beta-cell insulin secretion is closely coupled to alterations in ion channel conductances and intracellular Ca2+ ([Ca2+]i). To further examine this relationship after augmentation of voltage-dependent K+ channel expression, transgenic mice were produced which specifically overexpress a human insulinoma-derived, tetraethylammonium (TEA)-insensitive delayed rectifier K+ channel in their pancreatic beta-cells as shown by immunoblot of isolated islets and immunohistochemical analysis of pancreas sections. Whole-cell current recordings confirmed the presence of high amplitude TEA-resistant K+ currents in transgenic islet cells, whose expression correlated with hyperglycemia and hypoinsulinemia. Stable overexpression of the channel in insulinoma cells attenuated glucose-activated increases in [Ca2+]i and prevented the induction of TEA-dependent [Ca2+]i oscillations. These results, employing the first ion channel transgenic mouse, demonstrate the importance of membrane potential regulation in excitation-secretion coupling in the pancreatic beta-cell.

An insulin-responsive element in the pancreatic enhancer of the amylase gene.

We have identified a distinct insulin-responsive element (IRE) located within the pancreatic enhancer of the mouse amylase gene Amy-2.2. A 30-base pair (bp) fragment was previously shown to be sufficient to transfer insulin response to a heterologous promoter. The 30-bp fragment overlaps the tissue-specific pancreatic enhancer that binds the putative transcriptional activator PTF1. To determine whether enhancer and IRE activities could be separated, we introduced three 10-base pair substitutions into the 30-bp region. The mutated regulatory regions were cloned upstream of a heterologous promoter and transferred to transgenic mice. Mutants 1 and 2 retained PTF1 binding activity and insulin response. Mutant 3 retained PTF1 binding, but was defective in insulin response. The results indicate that the IRE is functionally distinct from the PTF1-binding site, although the two overlap physically. In contrast to the wild-type gene, mutant 3 is characterized by constitutive expression in diabetic animals, suggesting that a binding site for a repressor has been destroyed. A nuclear protein with affinity for the IRE was detected in normal and diabetic pancreas. This protein does not bind mutant 3, suggesting that it may be involved in negative regulation of amylase in diabetic pancreas.

Progressive hind limb paralysis in mice carrying a v-Mos transgene.

To study the function of the protooncogene Mos in mouse brain development we have created a transgenic mouse model system in which an activated form of the gene, the murine retroviral v-Mos gene, is highly overexpressed in the brain. Six transgenic founder animals and mice of one established transgenic line (line TG66) displayed a progressive hind limb paralysis with onset between 18 days and 9 months. The severity of the neurological phenotype correlated with pathological alterations and the degree of v-Mos expression in the brain which varied between individual animals of line TG66. The most striking feature of the brain pathology was the presence of large, abnormal astrocytes in the cerebellum, medulla, thalamus and in the dorsal horn of the spinal cord. These areas also contained shrunken and basophilic neurons whose cytoplasm was abnormally immunoreactive for phosphorylated epitopes of neurofilaments. In addition to neuropathologic changes, these mice also displayed aberrant eye lens differentiation and absence of hair cells in the inner ear. These results establish v-Mos transgenic mice as a model system to study progressive neurodegenerative disease and provide further evidence that the Mos protein-serine/threonine kinase has a function in brain development.

Endogenous retroviral sequences are required for tissue-specific expression of a human salivary amylase gene.

The human salivary amylase genes are associated with two inserted elements, a gamma-actin-processed pseudogene and an endogenous retroviral-like element. To test the contribution of these inserted elements to tissue specificity, 25 lines of transgenic mice carrying 10 amylase constructs were established. A 1-kb fragment of AMY1C (-1003 to +2) was found to be sufficient for parotid-specific expression of a human growth hormone reporter gene. The 1-kb fragment is entirely derived from inserted sequences. Deletion from -1003 to -826 resulted in reduced levels of transgene expression and loss of tissue specificity. The fragment -1003 to -327 was sufficient to transfer parotid specificity to the thymidine kinase promoter. The data demonstrate that the functional tissue-specific promoter of human AMY1C is derived from inserted sequences and that parotid expression can be conferred by sequences derived solely from the retrovirus. A role for retrotransposition in the evolution of gene regulation is indicated by these and other recent observations.

Harderian gland hyperplasia in c-mos transgenic mice.

Transgenic mice carrying the mouse mos proto-oncogene linked to a retroviral LTR develop hyperplasia of the Harderian glands. Enlargement of the glands is evident as early as 18 weeks after birth, with glands reaching up to 10 times their normal weight. Approximately 65% of the cases of hyperplasia occur bilaterally, and the majority of mice affected are male (66%). Elevated levels of mos expression are found in all Harderian glands of mice from the affected transgenic line, but not in glands of normal mice or a non-affected transgenic line, indicating that hyperplasia is dependent on mos expression. Histological examination of the tissue reveals a general involvement of the entire gland epithelium in hyperplastic growth, with no evidence of focal or malignant tumours. These observations show that in addition to neu, myc, ras and ret transgenes, mos, a member of the protein-serine/threonine kinase family of oncogenes, can induce Harderian gland hyperplasia, thus revealing an unusual response by this organ to various classes of oncogenes. Analysis of fos, jun, myc and ets oncogene RNA in mos-induced hyperplastic Harderian glands shows that there are no consistent changes in the level of expression of these oncogenes, suggesting that mos acts via a mechanism other than by increasing the expression of these genes.