Report of a family segregating mutations in both the <Emphasis Type="Italic">APC</Emphasis> and <Emphasis Type="Italic">MSH2</Emphasis> genes: juvenile onset of colorectal cancer in a double heterozygote

Background and aims  Mutations in DNA mismatch repair genes are associated with high risk of digestive malignancies [hereditary non-polyposis colorectal cancer (HNPCC); Lynch syndrome]; mutations of APC and MYH are associated with classic and attenuated familial adenomatous polyposis (FAP). Although the early onset of tumors in both syndromes is characteristic of their genetic origin, pediatric malignancies remain rare. Certain reports have found familial colorectal cancer (CRC) occurring in very young patients associated with mutations in more than one gene. Materials and method  A family corresponding to the Amsterdam criteria for HNPCC, including two cases of colorectal cancer before the age of 25 years, was analyzed for mutations in the MSH2 genes by sequencing. Because polyposis was observed in a patient who developed CRC at age 16, the APC gene was also sequenced. Results  A truncating mutation in the MSH2 gene, c.258_259delTG, was carried by patients developing cancer of the colon (two patients), uterus, kidney, bladder, and/or small intestine at ages 16, 24, 43, 44, 45, and 57, respectively. A patient with CRC at age 16 was found to carry the APC c.3183_3187del5 mutation as well as the MSH2 mutation, and it is inferred that her father, deceased of CRC at age 24, was also a double heterozygote. Interpretation  These results confirm that vigilance is required when interpreting molecular results for families with very young patients, as more than one gene may contribute to the genetic risk. Cancer screening measures must also be adapted to the earlier and more penetrant risk to double heterozygotes.     

Separation of killing and tumorigenic effects of an alkylating agent in mice defective in two of the DNA repair genes

Alkylation of DNA at the O⁶-position of guanine is one of the most critical events leading to mutation, cancer, and cell death. The enzyme O⁶-methylguanine-DNA methyltransferase repairs O⁶-methylguanine as well as a minor methylated base, O⁴-methylthymine, in DNA. Mouse lines deficient in the methyltransferase (MGMT) gene are hypersensitive to both the killing and to the tumorigenic effects of alkylating agents. We now show that these dual effects of an alkylating agent can be dissociated by introduction of an additional defect in mismatch repair. Mice with mutations in both alleles of the MGMT gene and one of the mismatch repair genes, MLH1, are as resistant to methylnitrosourea (MNU) as are wild-type mice, in terms of survival, but do have numerous tumors after receiving MNU. In contrast to MGMT^−/− MLH1^+/+ mice with decrease in size of the thymus and hypocellular bone marrow after MNU administration, no conspicuous change was found in MGMT^−/− MLH1^−/− mice treated in the same manner. Thus, killing and tumorigenic effects of an alkylating agent can be dissociated by preventing mismatch repair pathways.     

T cell functions in granulocyte/macrophage colony-stimulating factor deficient mice

Immunological functions were analyzed in mice lacking granulocyte/macrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to allo-antigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF +/+ and GM-CSF −/− mice. To investigate the responses of CD8⁺ and CD4⁺ T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD8⁺ T cells with specificity for ovalbumin peptide could not be induced in GM-CSF −/− mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF −/− mice. Purified CD4⁺ T cells from GM-CSF −/− mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-γ (IFN-γ) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD4⁺ T cells from GM-CSF −/− mice proliferated as well as those from GM-CSF +/+ mice and produced high amounts of IFN-γ and IL-4. To analyze the rescue effect of DC on CD4⁺ T cells, supernatants from (i) CD4⁺ T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD4⁺ T cells and KLH-pulsed spleen cells from GM-CSF −/− mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-γ production of CD4⁺ T cells from GM-CSF −/− mice.     

Inhibition of interferon γ induced interleukin 12 production: A potential mechanism for the anti-inflammatory activities of tumor necrosis factor

Inflammation is associated with production of cytokines and chemokines that recruit and activate inflammatory cells. Interleukin (IL) 12 produced by macrophages in response to various stimuli is a potent inducer of interferon (IFN) γ production. IFN-γ, in turn, markedly enhances IL-12 production. Although the immune response is typically self-limiting, the mechanisms involved are unclear. We demonstrate that IFN-γ inhibits production of chemokines (macrophage inflammatory proteins MIP-1α and MIP-1β). Furthermore, pre-exposure to tumor necrosis factor (TNF) inhibited IFN-γ priming for production of high levels of IL-12 by macrophages in vitro. Inhibition of IL-12 by TNF can be mediated by both IL-10-dependent and IL-10-independent mechanisms. To determine whether TNF inhibition of IFN-γ-induced IL-12 production contributed to the resolution of an inflammatory response in vivo, the response of TNF^+/+ and TNF^−/− mice injected with Corynebacterium parvum were compared. TNF^−/− mice developed a delayed, but vigorous, inflammatory response leading to death, whereas TNF^+/+ mice exhibited a prompt response that resolved. Serum IL-12 levels were elevated 3-fold in C. parvum-treated TNF^−/− mice compared with TNF^+/+ mice. Treatment with a neutralizing anti-IL-12 antibody led to resolution of the response to C. parvum in TNF^−/− mice. We conclude that the role of TNF in limiting the extent and duration of inflammatory responses in vivo involves its capacity to regulate macrophage IL-12 production. IFN-γ inhibition of chemokine production and inhibition of IFN-γ-induced IL-12 production by TNF provide potential mechanisms by which these cytokines can exert anti-inflammatory/repair function(s).     

Low density lipoprotein receptor-negative mice expressing human apolipoprotein B-100 develop complex atherosclerotic lesions on a chow diet: No accentuation by apolipoprotein(a)

We have generated mice with markedly elevated plasma levels of human low density lipoprotein (LDL) and reduced plasma levels of high density lipoprotein. These mice have no functional LDL receptors [LDLR^−/−] and express a human apolipoprotein B-100 (apoB) transgene [Tg(apoB^+/+)] with or without an apo(a) transgene [Tg(apoa^+/−)]. Twenty animals (10 males and 10 females) of each of the following four genotypes were maintained on a chow diet: (i) LDLR^−/−, (ii) LDLR^−/−;Tg(apoa^+/−), (iii) LDLR^−/−;Tg(apoB^+/+), and (iv)LDLR^−/−;Tg(apoB^+/+);Tg(apo^+/−). The mice were killed at 6 mo, and the percent area of the aortic intimal surface that stained positive for neutral lipid was quantified. Mean percent areas of lipid staining were not significantly different between the LDLR^−/− and LDLR^−/−;Tg(apoa^+/−) mice (1.0 ± 0.2% vs. 1.4 ± 0.3%). However, the LDLR^−/−;Tg(apoB^+/+) mice had ≈15-fold greater mean lesion area than the LDLR^−/− mice. No significant difference was found in percent lesion area in the LDLR^−/−;Tg(apoB^+/+) mice whether or not they expressed apo(a) [18.5 ± 2.5%, without lipoprotein(a), Lp(a), vs. 16.0 ± 1.7%, with Lp(a)]. Histochemical analyses of the sections from the proximal aorta of LDLR^−/−;Tg(apoB^+/+) mice revealed large, complex, lipid-laden atherosclerotic lesions that stained intensely with human apoB-100 antibodies. In mice expressing Lp(a), large amounts of apo(a) protein colocalized with apoB-100 in the lesions. We conclude that LDLR^−/−; Tg(apoB^+/+) mice exhibit accelerated atherosclerosis on a chow diet and thus provide an excellent animal model in which to study atherosclerosis. We found no evidence that apo(a) increased atherosclerosis in this animal model.     

Tumors of DNA mismatch repair-deficient hosts exhibit dramatic increases in genomic instability

DNA mismatch repair (MMR) deficiency is associated with an increased mutational burden and predisposition to certain malignancies. Relatively little is known, however, about gene-specific mutation frequencies within MMR-deficient primary tumors. Thymic lymphomas from Msh2^−/− mice were thus analyzed by using a lacI-based transgenic shuttle-phage mutation detection system. All tumors exhibited greatly elevated lacI gene mutation frequencies, ranging from 3.2- to 17.4-fold above the ≈15-fold elevations present within normal Msh2^−/− thymi. In addition, lacI genes harboring multiple changes, including clusters of mutations, were found in thymic tumor DNA. The results suggest that an additional mutator activity, such as an error-prone DNA polymerase, leads to increased genomic instability in these MMR-deficient tumors.     

Characterization of tumor necrosis factor-deficient mice

Although tumor necrosis factor (TNF) initially came to prominence because of its anti-tumor activity, most attention is now focused on its proinflammatory actions. TNF appears to play a critical role in both early and late events involved in inflammation, from localizing the noxious agent and amplifying the cellular and mediator responses at the local site and systemically, to editing (e.g., apoptosis) injured cells or effete immune cells and repairing inflammatory damage. We have generated mice deficient in TNF (TNF^−/− mice) and have begun to examine the multiple functions attributed to TNF. TNF^−/− mice develop normally and have no gross structural or morphological abnormalities. As predicted, they are highly susceptible to challenge with an infectious agent (Candida albicans), are resistant to the lethality of minute doses of lipopolysaccharide (LPS) following D-galactosamine treatment, have a deficiency in granuloma development, and do not form germinal centers after immunization. Phagocytic activity of macrophages appears relatively normal, as do T cell functions, as measured by proliferation, cytokine release, and cytotoxicity. B cell response to thymus-independent antigens is normal, but the Ig response to thymus-dependent antigen is reduced. Surprisingly, cytokine production induced by LPS appears essentially intact, with the exception of reduced colony-stimulating factor activity. Other unexpected findings coming from our initial analysis are as follows. (i) TNF has low toxicity in TNF^−/− mice. (ii) TNF^−/− mice show an anomalous late response to heat-killed Corynebacterium parvum. In contrast to the prompt response (granuloma formation, hepatosplenomegaly) and subsequent resolution phase in C. parvum-injected TNF^+/+ mice, similarly treated TNF^−/− mice show little or no initial response, but then develop a vigorous, disorganized inflammatory response leading to death. These results suggest that TNF has an essential homeostatic role in limiting the extent and duration of an inflammatory process—i.e., an anti-inflammatory function. (iii) In contrast to the expectation that TNF^+/+ mice and TNF^+/− mice would have identical phenotypes, TNF^+/− mice showed increased susceptibility to high-dose LPS lethality, increased susceptibility to Candida challenge, and delayed resolution of the C. parvum-induced inflammatory process, indicating a strong gene dose requirement for different actions of TNF.     

Morphology of sporadic colorectal cancer with DNA replication errors

Background—Up to 15% of colorectal cancers arecharacterised by DNA microsatellite instability (MIN), shown by thepresence of DNA replication errors (RERs).
Aims—To identify pathological features that arediscriminating for colorectal cancer (CRC) showing extensive MIN.
Subjects—A prospective series of 303 patientswith CRC and no family history of either familial adenomatous polyposisor hereditary non-polyposis colorectal cancer.
Methods—DNA was extracted from fresh tissuesamples and the presence of MIN was studied at nine loci that includedTGFβRII, IGFIIR, and BAX. The 61 cases showing RERs were comparedwith 63 RER negative cases with respect to a comprehensive set ofclinical and pathological variables. Predictive utility of thevariables was tested by decision tree analysis.
Results—Twenty seven patients with CRC showedextensive RERs (three loci or more) (RER+) and 34 had limited RERs only(28= one locus; 6 = two loci) (RER+/−), yielding a bimodaldistribution. RER+ cancers differed from RER− and RER+/− cases.Tumour type (adenocarcinoma, mucinous carcinoma, and undifferentiatedcarcinoma) (p=0.001), tumour infiltrating lymphocytes (p=0.001), andanatomical site (p=0.001) were the most significant of thediscriminating variables. Algorithms developed by decision treeanalysis allowed cases to be assigned to RER+ versus RER− and +/−status with a global sensitivity of 81.5%, specificity of 96%, andoverall accuracy of 93%.
Conclusion—Pathological examination of CRC allowsassignment of RER+ status; assignment is specific and relativelysensitive. Conversely RER− and RER+/− CRC are indistinguishable.

Keywords:colon; rectum; colorectal cancer; DNA replicationerrors; morphology; microsatellite instability

     

A novel <Emphasis Type="Italic">MSH2</Emphasis> mutation in a Chinese family with hereditary non-polyposis colorectal cancer

Background and aims Hereditary non-polyposis colorectal cancer (HNPCC) is one of the most common hereditary colon cancer syndrome accounting for 1–5% of all colorectal cancer cases. Germline mutations in DNA mismatch repair (MMR) genes are associated with the clinical phenotype of HNPCC. Defects in the MSH2 gene may account for about 40% of HNPCC cases including nucleotide substitutions, deletions and insertions. Only a few mutations were reported in Chinese families. This study was performed to identify the disease-causing gene mutation(s) and conduct pre-symptomatic diagnosis in a Chinese family with HNPCC. Materials and methods Mutation analysis in MLH1 and MSH2 followed by pre-symptomatic diagnosis in MSH2 was performed on genomic DNA isolated from the family members. Results A novel duplication mutation of four nucleotides in exon 7 of MSH2 (MSH2: c.1216_1219dupCGAC) resulting in a premature stop 10 codons downstream in MSH2 (p.L407fsX417) was found. The mutation was associated with HNPCC and an asymptomatic carrier was found in the family. Conclusion HNPCC in a Chinese family is associated with a novel mutation in the MSH2 gene (MSH2: c.1216_1219dupCGAC). Duo Zheng, Tiegang Li and Xiaoping Liu have contributed equally to this work.     

Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5′ CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI− tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2′-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.     

Gene targeting by linear duplex DNA frequently occurs by assimilation of a single strand that is subject to preferential mismatch correction

To study targeted recombination, a single linear 2-kb fragment of LEU2 DNA was liberated from a chromosomal site within the nucleus of Saccharomyces cerevisiae, by expression of the site-specific HO endonuclease. Gene targeting was scored by gene conversion of a chromosomal leu2 mutant allele by the liberated LEU2 fragment. This occurred at a frequency of only 2 × 10⁻⁴, despite the fact that nearly all cells successfully repaired, by single-strand annealing, the chromosome break created by liberating the fragment. The frequency of Leu⁺ recombinants was 6- to 25-fold higher in pms1 strains lacking mismatch repair. In 70% of these cases, the colony was sectored for Leu⁺/Leu⁻. Similar results were obtained when a 4.1-kb fragment containing adjacent LEU2 and ADE1 genes was liberated, to convert adjacent leu2 and ade1 mutations on the chromosome. These results suggest that a linear fragment is not assimilated into the recipient chromosome by two crossovers each close to the end of the fragment; rather, heteroduplex DNA between the fragment and the chromosome is apparently formed over the entire region, by the assimilation of one of the two strands of the linear duplex DNA. Moreover, the recovery of Leu⁺ transformants is frequently defeated by the cell’s mismatch repair machinery; more than 85% of mismatches in heteroduplex DNA are corrected in favor of the resident, unbroken (mutant) strand.     

Variable and tissue-specific hormone resistance in heterotrimeric Gs protein α-subunit (Gsα) knockout mice is due to tissue-specific imprinting of the Gsα gene

Albright hereditary osteodystrophy (AHO), an autosomal dominant disorder characterized by short stature, obesity, and skeletal defects, is associated with heterozygous inactivating mutations of GNAS1, the gene encoding the heterotrimeric G protein α-subunit (G_sα) that couples multiple receptors to the stimulation of adenylyl cyclase. It has remained unclear why only some AHO patients present with multihormone resistance and why AHO patients demonstrate resistance to some hormones [e.g., parathyroid hormone (PTH)] but not to others (e.g., vasopressin), even though all activate adenylyl cyclase. We generated mice with a null allele of the mouse homolog Gnas. Homozygous G_s deficiency is embryonically lethal. Heterozygotes with maternal (m−/+) and paternal (+/p−) inheritance of the Gnas null allele have distinct phenotypes, suggesting that Gnas is an imprinted gene. PTH resistance is present in m−/+, but not +/p−, mice. G_sα expression in the renal cortex (the site of PTH action) is markedly reduced in m−/+ but not in +/p− mice, demonstrating that the Gnas paternal allele is imprinted in this tissue. Gnas is also imprinted in brown and white adipose tissue. The maximal physiological response to vasopressin (urinary concentrating ability) is normal in both m−/+ and +/p− mice and Gnas is not imprinted in the renal inner medulla (the site of vasopressin action). Tissue-specific imprinting of Gnas is likely the mechanism for variable and tissue-specific hormone resistance in these mice and a similar mechanism might explain the variable phenotype in AHO.     

Targeted disruption of the Ca2+ channel β3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltagedependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca²⁺ channels, the pore-forming, antagonist-binding α₁ subunit, considerably less is understood about how β subunits contribute to neuronal Ca²⁺ channel function. We studied the role of the Ca²⁺ channel β₃ subunit, the major Ca²⁺ channel β subunit in neurons, by using a gene-targeting strategy. The β₃ deficient (β₃−/−) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic β₃−/− neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca²⁺ channels was described by two Boltzmann components with different voltage dependence, analogous to the “reluctant” and “willing” states reported for N-type channels. The absence of the β₃ subunit was associated with a hyperpolarizing shift of the “reluctant” component of activation. Norepinephrine inhibited wt and β₃−/− neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca²⁺ channels. The reduction in the expression of N-type Ca²⁺ channels in the β₃−/− mice may be expected to impair Ca²⁺ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

Nonmuscle myosin II-B is required for normal development of the mouse heart

We used targeted gene disruption in mice to ablate nonmuscle myosin heavy chain B (NMHC-B), one of the two isoforms of nonmuscle myosin II present in all vertebrate cells. Approximately 65% of the NMHC-B^−/− embryos died prior to birth, and those that were born suffered from congestive heart failure and died during the first day. No abnormalities were detected in NMHC-B^+/− mice. The absence of NMHC-B resulted in a significant increase in the transverse diameters of the cardiac myocytes from 7.8 ± 1.8 μm (right ventricle) and 7.8 ± 1.3 μm (left ventricle) in NMHC-B^+/+ and B^+/− mice to 14.7 ± 1.1 μm and 13.8 ± 2.3 μm, respectively, in NMHC-B^−/− mice (in both cases, P < 0.001). The increase in size of the cardiac myocytes was seen as early as embryonic day 12.5 (4.5 ± 0.2 μm for NMHC-B^+/+ and B^+/− vs. 7.2 ± 0.6 μm for NMHC-B^−/− mice (P < 0.01)). Six of seven NMHC-B^−/− newborn mice analyzed by serial sectioning also showed structural cardiac defects, including a ventricular septal defect, an aortic root that either straddled the defect or originated from the right ventricle, and muscular obstruction to right ventricular outflow. Some of the hearts of NMHC-B^−/− mice showed evidence for up-regulation of NMHC-A protein. These studies suggest that nonmuscle myosin II-B is required for normal cardiac myocyte development and that its absence results in structural defects resembling, in part, two common human congenital heart diseases, tetralogy of Fallot and double outlet right ventricle.     

Alkylation of porcine nucleic acids following intravenous and intragastric application of N-methyl-N-nitrosourea

Summary Alkylation of nucleic acids was measured 3 h after the administration of N-(¹⁴C)-methyl-N-nitrosourea (10 mg/kg) to Hanford mini pigs. After intravenous injection the amount of 7-methylguanine formed in DNA and RNA of liver, kidney, intestines, and brain, was 2–3 times higher than after administration of a similar dose by stomach tube. In the stomach, however, the level of alkylation was more than 8 times higher after intragastric application when compared to intravenous injection. In vitro studies on the stability of N-methyl-N-nitrosourea in gastric juice indicate that no significant decomposition of the carcinogen occurred within the gastric lumen. This suggests that after application by stomach tube a substantial proportion of N-methyl-N-nitrosourea reacts with stomach cells during resorption. The results are discussed with respect to current investigations on the carcinogenic effect of N-methyl-N-nitrosourea in this species.

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Alkylierung der Nucleinsäuren von Hanford mini pigs nach intravenöser und intragastrischer Applikation von N-Methyl-N-nitroso-Harnstoff

Zusammenfassung Die Alkylierung von Nucleinsäuren in vivo wurde bestimmt 3 Stunden nach Gabe einer einmaligen Dosis von N(¹⁴C)Methyl-N-Nitrosoharnstoff (10 mg/kg) an Hanford mini pigs. Nach intravenöser Injektion fand sich in der DNA und RNA von Leber, Niere, Dünndarm und Gehirn 2–3mal mehr 7-Methylguanin als nach Applikation mit der Schlundsonde. Allerdings war nach intragastrischer Applikation die Alkylierung der Nucleinsäuren des Magens mehr als 8mal höher als bei intravenöser Injektion. Bei in vitro-Untersuchungen ergab sich kein Hinweis für einen nennenswerten Zerfall des Methylnitrosoharnstoff innerhalb des Magenlumens. Es wird deshalb angenommen, daß nach Applikation mit der Schlundsonde ein beträchtlicher Teil des Cancerogens während der Resorption mit den Zellen der Magenwand reagiert. Die Ergebnisse werden diskutiert im Hinblick auf laufende Untersuchungen zur cancerogenen Wirkung von Methylnitrosoharnstoff an Hanford mini pigs.

     

Thymocyte development is normal in CTLA-4-deficient mice

Recent studies indicate that CTLA-4 interaction with B7 ligands transduces an inhibitory signal to T lymphocytes. Mice homozygous for a null mutation in CTLA-4 have provided the most dramatic example of the functional importance of CTLA-4 in vivo. These animals develop a fatal lymphoproliferative disorder and were reported to have an increase in CD4⁺ and CD8⁺ thymocytes and CD4⁻CD8⁻ thymocytes, and a decrease in CD4⁺CD8⁺ thymocytes. Based on these observations, it was proposed that CTLA-4 is necessary for normal thymocyte development. In this study, CTLA-4-deficient mice carrying an insertional mutation into exon 3 of the ctla-4 gene were generated. Although these mice display a lymphoproliferative disorder similar to previous reports, there was no alteration in the thymocyte profiles when the parathymic lymph nodes were excluded from the thymi. Further, thymocyte development was normal throughout ontogeny and in neonates, and there was no increase in thymocyte production. Finally, T cell antigen receptor signaling, as assessed by proximal and distal events, was not altered in thymocytes from CTLA-4^−/− animals. Collectively, these results clearly demonstrate that the abnormal T cell expansion in the CTLA-4-deficient mice is not due to altered thymocyte development and suggest that the apparent altered thymic phenotype previously described was due to the inclusion of parathymic lymph nodes and, in visibly ill animals, to the infiltration of the thymus by activated peripheral T cells. Thus it appears that CTLA-4 is primarily involved in the regulation of peripheral T cell activation.     

Escherichia coli DNA polymerase II catalyzes chromosomal and episomal DNA synthesis in vivo

We have investigated a role for Escherichia coli DNA polymerase II (Pol II) in copying chromosomal and episomal DNA in dividing cells in vivo. Forward mutation frequencies and rates were measured at two chromosomal loci, rpoB and gyrA, and base substitution and frameshift mutation frequencies were measured on an F′(lacZ) episome. To amplify any differences in polymerase error rates, methyl-directed mismatch repair was inactivated. When wild-type Pol II (polB⁺) was replaced on the chromosome by a proofreading-defective Pol II exo⁻ (polBex1), there was a significant increase in mutation frequencies to rifampicin resistance (Rif^R) (rpoB) and nalidixic acid resistance (Nal^R) (gyrA). This increased mutagenesis occurred in the presence of an antimutator allele of E. coli DNA polymerase III (Pol III) (dnaE915), but not in the presence of wild-type Pol III (dnaE⁺), suggesting that Pol II can compete effectively with DnaE915 but not with DnaE⁺. Sequencing the Rif^R mutants revealed a G → A hot spot highly specific to Pol II exo⁻. Pol II exo⁻ caused a significant increase in the frequency of base substitution and frameshift mutations on F′ episomes, even in dnaE⁺ cells, suggesting that Pol II is able to compete with Pol III for DNA synthesis on F episomes.     

Shortage of mitogen-activated protein kinase is responsible for resistance to AP-1 transactivation and transformation in mouse JB6 cells

The JB6 mouse epidermal cell system, which includes tumor promotion-sensitive (P⁺) and tumor promotion-resistant (P⁻) cells, is a well-established and extensively used cell culture model for studying the mechanism of late-stage tumor promotion. Tumor promoters, such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or epidermal growth factor (EGF), induce high levels of activator protein 1 (AP-1) activity and large, tumorigenic, anchorage-independent colonies in soft agar at a high frequency in JB6 P⁺ cells, but not in JB6 P⁻ cells. We report here a molecular explanation for the defect in the AP-1 activation and promotion-resistant phenotype of P⁻ cells. We demonstrate that the lack of AP-1 activation and cell transformation responses to TPA and EGF in P⁻ cells appears attributable to the low level of mitogen-activated protein kinase (MAPK) (extracellular signal-regulated protein kinase, Erk) in these cells. TPA and EGF induce transactivation of AP-1 activity in P⁺ cells but not in P⁻ cells. Nonphosphorylated forms and TPA- or EGF-induced phosphorylated forms of Erks (Erk1 and Erk2) in P⁻ cells were much lower than those in P⁺ cells. Stable transfection of wild-type MAPK (Erk2) into P⁻ cells restored its response to TPA and EGF for both AP-1 activation and cell transformation. These results suggest that the shortage of MAPK (Erk1 and Erk2) appears to be an important contributor to the tumor promotion-resistant phenotype in JB6 cells.     

Monoaminergic control of MSH release in the lizard Anolis carolinensis

The release of MSH, in vivo or in vitro, was investigated in the lizard Anolis carolinensis. A single intraperitoneal (ip) injection of α-methyl-p-tyrosine (400 μg/g body wt) produced an intense and long-lasting darkening response in animals adapted to a white illuminated background; α-methyl-m-tyrosine (400 μg/g body wt) and α-methyl-DOPA (100 μg/g body wt) also produced a darkening response, but the effect was slighter and shorter. Pimozide (10 μg/g body wt) also gave rise to a darkening response and para-chlorophenylalanine (400 μg/g body wt) completely abolished the darkening produced by pimozide or α-methyl-p-tyrosine. Hypophysectomized animals did not change from green color when injected with any of the darkening drugs. Incubation of neurointermediate halves with serotonin (5.2 × 10^?7M), dopamine (2.6 × 10^?6M), or serotonin plus dopamine showed that dopamine appreciably inhibits the serotonin-stimulated release of this hormone. These results suggest that a serotoninergic stimulatory, and a dopaminergic inhibitory, mechanism controls the release of MSH from the pars intermedia of the Anolis pituitary gland.     

Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities

Npt2 encodes a renal-specific, brush-border membrane Na⁺-phosphate (P_i) cotransporter that is expressed in the proximal tubule where the bulk of filtered P_i is reabsorbed. Mice deficient in the Npt2 gene were generated by targeted mutagenesis to define the role of Npt2 in the overall maintenance of P_i homeostasis, determine its impact on skeletal development, and clarify its relationship to autosomal disorders of renal P_i reabsorption in humans. Homozygous mutants (Npt2^−/−) exhibit increased urinary P_i excretion, hypophosphatemia, an appropriate elevation in the serum concentration of 1,25-dihydroxyvitamin D with attendant hypercalcemia, hypercalciuria and decreased serum parathyroid hormone levels, and increased serum alkaline phosphatase activity. These biochemical features are typical of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a Mendelian disorder of renal P_i reabsorption. However, unlike HHRH patients, Npt2^−/− mice do not have rickets or osteomalacia. At weaning, Npt2^−/− mice have poorly developed trabecular bone and retarded secondary ossification, but, with increasing age, there is a dramatic reversal and eventual overcompensation of the skeletal phenotype. Our findings demonstrate that Npt2 is a major regulator of P_i homeostasis and necessary for normal skeletal development.