Sequence-dependent DNA replication in preimplantation mouse embryos.

Circular, double-stranded DNA molecules were injected into nuclei of mouse oocytes and one- or two-cell embryos to determine whether specific sequences were required to replicate DNA during mouse development. Although all of the injected DNAs were stable, replication of plasmid pML-1 DNA was not detected unless it contained either polyomavirus (PyV) or simian virus 40 (SV40) DNA sequences. Replication occurred in embryos, but not in oocytes. PyV DNA, either alone or recombined with pML-1, underwent multiple rounds of replication to produce superhelical and relaxed circular monomers after injection into one- or two-cell embryos. SV40 DNA also replicated, but only 3% as well as PyV DNA. Coinjection of PyV DNA with either pML-1 or SV40 had no effect on the replicating properties of the three DNAs. These results are consistent with a requirement for specific cis-acting sequences to replicate DNA in mammalian embryos, in contrast to sequence-independent replication of DNA injected into Xenopus eggs. Furthermore, PyV DNA replication in mouse embryos required PyV large T-antigen and either the alpha-beta-core or beta-core configuration of the PyV origin of replication. Although the alpha-core configuration replicated in differentiated mouse cells, it failed to replicate in mouse embryos, demonstrating cell-specific activation of an origin of replication. Replication or expression of PyV DNA interfered with normal embryonic development. These results reveal that mouse embryos are permissive for PyV DNA replication, in contrast to the absence of PyV DNA replication and gene expression in mouse embryonal carcinoma cells.

Microinjection of metallothionein-oncomodulin DNA into fertilized mouse embryos is correlated with fetal lethality.

Oncomodulin (ONCO) is an oncodevelopmental protein expressed in placental and extraembryonic tissue and re-expressed in a wide variety of tumors. The metallothionein promoter (MT) is active in numerous adult tissues, in parietal and visceral extraembryonic endoderm, and developing liver. To study the function of oncomodulin we microinjected MT-ONCO DNA into one-cell embryos and examined tissues of fetal and adult mice. Analysis of implant sites from embryos, microinjected with MT-ONCO DNA then placed into pseudopregnant females, indicated a greater than three-fold increase in empty and necrotic implant sites relative to SV2NEO-microinjected embryos and a seven-fold rise relative to non-microinjected embryos. The striking feature of the lethality was the presence of a normal placenta but absence of fetal tissue. Few MT-ONCO DNA transgenic mice were isolated (3.5%) and none were able to express oncomodulin protein or RNA in any tissue examined, even after prolonged heavy metal stimulation of the MT promoter. Fetal mortality is best correlated with expression of oncomodulin causing an interruption of either cellular differentiation or organogenesis before day 9 in development.

Polyomavirus large T-antigen expression in heart of transgenic mice causes cardiomyopathy.

One of the early genes of polyomavirus, large T-antigen (PVLT), has been classified in vitro as an immortalizing gene. In order to determine the ability of PVLT to cause the formation of hyperplasia or tumors in vivo, we generated transgenic mice harboring the cDNA for PVLT linked to the heavy-metal responsive metallothionein-1 promoter (MT). The transgene was primarily expressed in testes and seminal vesicles, but expression was also detected in heart of a single transgenic line. The expression of the transgene in the heart of MT-PVLT line 8 mice was correlated with cardiomyopathy and atrial thrombus formation leading to premature death at approximately 160 days due to cardiac failure. The heart of affected animals was from 1.5 to 5.2 fold greater in weight and 2 fold greater in dimensions than normal nontransgenic mice. Affected hearts fell short of frank tumor phenotype and no macroscopic nor microscopic focal growth was found. Histologically the heart has a heterogenous cardiomyocyte population with markedly enlarged cells mixed with relatively normal cells. Both cell types express PVLT protein. The primary cell type affected is the cardiomyocyte however, as heart proportions are maintained, interstitial and non-myocyte cells must be affected either directly or indirectly. Expression of PVLT has upset normal strict control of cell growth in these hearts to result in a new model of congestive cardiomyopathy.

The characterization of the uptake of avidin-biotin complex by HeLa cells.

HeLa cells have been shown to internalize the avidin-biotin complex. Adsorptive pinocytosis seems to be the mechanism of this uptake as seen by the requirements of energy and the integrity of the microtubular assembly. Pretreatment of HeLa cells with cycloheximide inhibits uptake and binding of the avidin-biotin complex. Scatchard plot of specific binding of avidin indicates a single type of binding with a Kd of approx. 55 pM with about 21,500 receptors/cell. The lack of inhibition of binding by simple carbohydrates indicates that binding is not through the oligosaccharide chain of avidin.

Cloning and characterization of a novel gene that is regulated by estrogen and is associated with mammary gland carcinogenesis.

Estrogens play a role in mammary gland function and are implicated in mammary carcinogenesis. We report the cloning of a novel gene [steroid-sensitive gene 1 (SSG1)] that is regulated by E(2) in the rat uterus and mammary gland. The full-length SSG1 complementary DNA has an open reading frame of 1158 nucleotides encoding a putative protein of 385 amino acids. A SSG1-specific antibody recognizes a 40-kDa protein localized to myoepithelial cells of normal mammary tissue and to endothelial cells of 7,12-dimethylbenz(a)antracene-induced mammary tumors. Treatment of rats with E(2) at 1.2 or 2.4 microg/kg.day for 21 days increases SSG1 protein levels in mammary tissue by 16-fold compared with controls. Removal of E(2) after a 14-day treatment decreases SSG1 protein levels 6-fold and 3-fold at 120 and 144 h, respectively. Treatment of rats with the estrogen antagonists tamoxifen or ICI 182,780 did not affect SSG1 protein levels compared with controls. SSG1 protein levels in 7,12-dimethylbenz(a)antracene-induced rat mammary tumors were 23-fold greater than SSG1 levels in resting mammary tissue, and 8-fold higher than protein levels expressed in lactating mammary glands. We propose that SSG1 plays a role in estrogen functions, and its overexpression is correlated with mammary carcinogenesis.

Steroid-sensitive gene-1 is an androgen-regulated gene expressed in prostatic smooth muscle cells in vivo.

Steroid-sensitive gene-1 (SSG1) is a novel gene we cloned, found regulated by 17beta-estradiol in the rat uterus and mammary gland, and over-expressed in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. We show here that SSG1 mRNA and protein expression are regulated by androgens in the rat ventral prostate. Increases in SSG1 mRNA levels were detected by Northern blotting after 24 h and reached a 27-fold peak 96 h following castration, relative to SSG1 mRNA expression in sham-operated rats. Dihydrotestosterone or testosterone supplementation of castrated rats prevented this rise in SSG1 mRNA. In contrast with SSG1 mRNA expression, SSG1 protein was decreased 16-fold 2 weeks following castration but was at control levels in the prostates of castrated rats receiving dihydrotestosterone or testosterone. Although SSG1 is regulated by androgens in vivo, treatment of LnCap cells with dihydrotestosterone, cyproterone acetate or flutamide did not result in the regulation of SSG1 protein levels in vitro. Immunofluorescence studies show that SSG1 is mainly expressed in prostatic smooth muscle cells. These results indicate that SSG1 is an androgen-regulated gene that is expressed in the smooth muscle component of the rat ventral prostate in vivo.

Expression of immediate early genes, GATA-4, and Nkx-2.5 in adrenergic-induced cardiac hypertrophy and during regression in adult mice.

Adrenoreceptor agonists induce a hypertrophic phenotype in vitro and in vivo. To investigate the molecular remodeling in chronic cardiac hypertrophy we infused adult male mice with vehicle. isoproterenol, phenylephrine or both agonists for 3, 7 or 14 days. All drugs increased cardiac mass. After minipump removal cardiac mass regressed to control levels within 7 days after PE and ISO treatment whereas ISO + PE treated hearts were incompletely regressed. ANF and beta-MHC, but not alpha-MHC, expression were increased by agonists at all time points. GATA-4, Nkx-2.5, Egr-1, c-jun and c-fos expression were increased after 3, 7 and 14 days of treatment. Expression was greatest after ISO+PE> >ISO>PE>vehicle infusion suggesting a synergistic effect of adrenoreceptor stimulation and indicating a greater effect of beta- than alpha-adrenergic action in vivo. After PE or ISO drug withdrawal the HW/BW was normal and Egr-1, c-jun, c-fos and GATA-4, but not Nkx2.5, expression dropped to control levels. HW/BW regression was incomplete after ISO+PE and elevated levels of Egr-1, c-jun and Nkx2.5 expression remained. A hydralazine-mediated reduction in blood pressure had no effect on the agonist-induced cardiac hypertrophy or gene expression. In conclusion, we found that continued agonist stimulation, and not blood pressure. is responsible for the maintained increase in gene expression. Further, we found the decrease in gene expression in the regression after drug withdrawal was gene specific.

Differential effects of cysteamine on heat shock protein induction and cytoplasmic granulation in astrocytes and glioma cells.

The sulfhydryl agent, cysteamine (CSH), promotes the accumulation of autofluorescent, peroxidase-positive cytoplasmic granules in cultured astroglia akin to those which naturally accumulate in astrocytes of the aging periventricular brain. Both in vitro and in situ, CSH rapidly induces various heat shock proteins (HSP) in astrocytes long before granulation occurs. In the present study, we determined that CSH treatment resulted in an increase in HSP 27, HSP 90 and heme oxygenase (HO-1) at both the protein and mRNA level. We also showed that C6 glioma cells, unlike primary astrocytes, constitutively express HSP 27, HSP 90 and HO-1 at low levels. Moreover, CSH is incapable of eliciting further HSP expression or inducing granulation in the glioma cells. Our results support the hypothesis that the biogenesis of redox-active astrocytic inclusions in CSH-treated glial cultures and in the aging periventricular brain is dependent on an antecedent cellular stress response.

Analysis of gene expression during experimental uveitis in the rabbit.

OBJECTIVE: To analyse the pattern of gene expression at the messenger RNA level during ocular inflammation in the rabbit. DESIGN: A gene screen assay was used to quantify specific binding over background (expression index) of various activation markers and cytokines in rabbit iris-ciliary body obtained during active experimental uveitis induced by injection of porcine lens protein (two animals) and in a control group (two animals). OUTCOME MEASURES: Expression indices corresponding to the activation markers and cytokines assayed. RESULTS: Compared with the control eyes, analysis of triplicate samples from the inflamed eyes showed a significantly higher expression index corresponding to the proto-oncogenes c-fos, c-jun and Ha-ras, interleukin-2 and heat shock protein Hsp27 and a significantly lower index corresponding to transforming growth factor-beta (TGF-beta) (p < 0.05). CONCLUSIONS: Active experimental lens-induced uveitis is associated with a significant rise in the gene expression of cellular activation factors and a decrease in an immunoprotective factor (TGF-beta) in the iris and ciliary body of the rabbit.

Effect of dexrazoxane and amifostine on the vertebral bone quality of Doxorubicin treated male rats.

Doxorubicin (DOX) is widely used in combination cocktails for treatment of childhood hematological cancers and solid tumors. A major factor limiting DOX usage is DOX-induced cardiotoxicity. However, it is not known whether protectants like dexrazoxane (DXR) and amifostine (AMF) can prevent DOX-mediated bone damage. The present study investigated whether administration of AMF alone or in combination with DXR would prevent any DOX-mediated bone damage. Male rat pups were treated with DOX, DXR, AMF, and their combinations. On neonate day 38, the bone mineral density (BMD), bone mineral content (BMC) and the micro-architecture of the lumbar vertebrae were analyzed. We have shown that when male rats are treated with DOX, DXR, DOX+DXR, AMF, DOX+AMF or DOX+DXR+AMF, there is a decrease in lumbar vertebral BMD (p<0.05). Furthermore, the relative bone volume (BV/TV) was decreased by DXR, DOX+DXR, and DOX+AMF treatments. Interestingly, DOX+AMF significantly increased BV/TV when compared to DXR treatment (p<0.04). The trabecular number (Tb.N) decreased with DXR and DOX+DXR and increased with DOX+AMF treatments. This information will be useful in designing better cancer combination therapies that do not lead to vertebrae deterioration.

Gender-dependent reductions in vertebrae length, bone mineral density and content by doxorubicin are not reduced by dexrazoxane in young rats: effect on growth plate and intervertebral discs.

Doxorubicin (DOX) is widely used in anti-cancer cocktails. Dexrazoxane (DXR) is a cardioprotectant approved for use with DOX. The effect of DOX, with or without DXR, on bone in children is not well understood. The aim of this study was to examine the effect of DOX on vertebrae and femur length and bone density acquisition in young rats, as well as to test the hypothesis that young females are more susceptible to DOX-induced tissue damage than young males. The results of this study suggest that a single injection of DOX in young female and not male rats is associated with low bone turnover resulting in vertebrae and femur bone growth deficits. DOX selectively decreased BMD and BMC accrual in the lumbar vertebrae that was not prevented by DXR. DOX-treated rats also exhibited growth plate and intervertebral disc defects. This information will be useful in the design of interventions to promote bone growth or retard bone loss during DOX treatment.

Amifostine and dexrazoxane enhance the rapid loss of bone mass and further deterioration of vertebrae architecture in female rats.

Doxorubicin (DOX) is widely used in combination cocktails for treatment of childhood hematologic cancers and solid tumors. A major factor limiting DOX usage is DOX-induced cardiotoxicity. Dexrazoxane (DXR) is an iron-binding compound and the only approved cardioprotectant for use with DOX. Amifostine (AMF) is a free radical scavenger and approved as a broad-spectrum cytoprotectant. We have shown that when female rats are treated with AMF, AMF + DOX, or AMF + DXR + DOX there is a significant decrease in the right femoral and lumbar vertebral bone mineral density (BMD) (P < 0.05) but not in the left femoral BMD. Furthermore, the relative bone volume (BV/TV) was significantly smaller in the lumbar vertebral bodies of rats treated with AMF (21.1%), AMF + DOX (34.4%), and AMF + DXR + DOX (38.4%), as was the trabecular number (Tb.N) with AMF (15.5%), AMF + DOX (29.9%), and AMF + DXR + DOX (32.3%). AMF + DOX- and AMF + DXR + DOX-treated vertebrae also exhibited deterioration in the microarchitecture of the trabecular bone and spinous processes as ascertained by microcomputerized tomography (micro CT). This information will be useful in designing better cancer combination therapies that do not lead to bone deterioration.

The biotin requirement of HeLa cells.

We have examined the effect of alterations in the biotin content of the medium on the growth, viability, biotin content, and the activities of biotin-independent and biotin-independent enzymes of the HeLa cells. The inclusion in the growth medium of avidin, which almost irreversibly binds with biotin (Kd, 10(-15) M), results in an increase in cellular biotin content and biotin enzyme activity over that seen when the cells are grown in a biotin-depleted medium. The addition of avidin-bound biotin to the growth medium led to a forty-fold increase in cellular biotin when compared to the inclusion of an equivalent amount of free biotin in the medium. HeLa cells are able to internalize avidin-bound biotin. Biotin is released from this complex to function as the prosthetic group of biotin enzymes. HeLa cells do have a nutritional requirement for biotin.

The partial characterization of the binding of avidin-biotin complex to rat liver plasma membrane.

Rat liver plasma membrane and rat intestinal mucosa plasma membrane were examined for their ability to bind avidin-[3H]biotin complex. Avidin-[3H]biotin complex bound specifically to liver plasma membrane with a Kd of approx. 35 nM and Bmax. of 136 pmol/mg of membrane protein. The receptor on the liver plasma membrane was exposed by Pronase, sensitive to tryptic hydrolysis and insensitive to neuraminidase. The optimum pH of specific binding was less than 6.0. Simple carbohydrates, mannan and ovalbumin did not inhibit specific binding. Periodate-treated or alpha-mannosidase-treated avidin was able to inhibit binding of either avidin-[3H]biotin complex or untreated avidin to plasma membrane. [3H]Avidin containing methylated lysine residues did not bind to rat liver plasma membrane. There was no specific binding of the avidin-biotin complex to rat intestinal mucosa plasma membrane.

Immortal cell lines isolated from heart differentiate to an endothelial cell lineage in the presence of retinoic acid.

Previously, we isolated a single line of transgenic mice which develop an enlarged heart due to the expression of the immortalizing gene, polyomavirus large T antigen. Immortal cell lines were isolated from adult transgenic but not from nontransgenic hearts. All of the 24 cell lines expressed vimentin and fibronectin but not desmin or myosin heavy chain. We conclude that the cell lines are of non-muscle origin. Six cell lines were chosen for further study. All six cell lines demonstrate profound morphological and biochemical effects when incubated with 10(-4) M to 10(-7) M retinoic acid. The retinoic acid-treated cell lines showed arrested cellular proliferation and aligned to form rows and vesicle-like structures. Cycloheximide inhibited these retinoic acid-induced changes, indicating a need for continued protein synthesis. Retinoic acid-treated, but not untreated, cells lost expression of vimentin and fibronectin, gained the ability to incorporate acetylated low density lipoprotein, and expressed Factor VIII-related antigen. Retinoic acid did not induce expression of desmin or myosin heavy chain. Incubation of the cell lines with transforming growth factor beta 1, dimethyl sulfoxide, or phorbol esters had no biochemical or morphological effect. We conclude that these cell lines differentiate to an endothelial lineage in the presence of retinoic acid.

Altered molecular response to adrenoreceptor-induced cardiac hypertrophy in Egr-1-deficient mice.

Unmanipulated early growth response-1 (Egr-1)-deficient -/- mice have similar heart-to-body weight ratios but express lower amounts of atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), skeletal actin, NGF1-A binding protein (NAB)-2, Sp1, c-fos, c-jun, GATA-4, and Nkx2.5 than +/+ or +/- mice. alpha-MHC, tubulin, and NAB-1 expression was similar. Isoproterenol (Iso) and phenylephrine (PE) infusion into +/+ and -/- mice increased heart weight, ANF, beta-MHC, skeletal actin, Sp1, NAB-2, c-fos, and c-jun expression, but induction in -/- mice was lower. Only Iso + PE-treated +/+ mice showed induction of NAB-1, GATA-4, and Nkx2.5. Foci of fibrosis were found in Iso + PE-treated -/- and +/+ mice. Surprisingly, vehicle-treated -/- mice displayed fibrosis and increased Sp1, skeletal actin, Nkx2.5, and GATA-4 expression without hypertrophy. Minipump removal caused the agonist-treated hearts and gene expression to regress to control or near-control levels. Thus Egr-1 deficiency caused a blunted catecholamine-induced hypertrophy response and increased sensitivity to stress.

TAFII250, Egr-1, and D-type cyclin expression in mice and neonatal rat cardiomyocytes treated with doxorubicin.

Differential display identified that gene fragment HA220 homologous to the transcriptional activator factor II 250 (TAFII250) gene, or CCG1, was increased in hypertrophied rodent heart. To determine whether TAFII250 gene expression is modified after cardiac damage, we measured TAFII250 expression in vivo in mouse hearts after injection of the cardiotoxic agent doxorubicin (DXR) and in vitro in DXR-treated isolated rat neonatal cardiomyocytes. In vivo atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), Egr-1, and TAFII250 expression increased with dose and time after a single DXR injection, but only ANF and beta-MHC expression were increased after multiple injections. After DXR treatment of neonatal cardiomyocytes we found decreased ANF, alpha-MHC, Egr-1, and TAFII250 expression. Expression of the TAFII250-regulated genes, the D-type cyclins, was increased after a single injection in adult mice and was decreased in DXR-treated cardiomyocytes. Thus expression of Erg-1, TAFII250, and the D-type cyclins is modulated after cardiotoxic damage in adult and neonatal heart.

cis- and trans-acting sequences required for expression of simian virus 40 genes in mouse oocytes.

To determine the requirements for gene expression in mammalian germ cells, circular double-stranded simian virus 40 (SV40) DNA molecules containing deletions in sequences controlling transcription and replication were injected into the nucleus of mouse oocytes. Expression of large (T-Ag) and small (t-Ag) tumor antigens ("early gene products") required at least three GGGCGG boxes, but did not require either the origin of viral DNA replication (ori) or a TATA box. Expression of capsid antigen VP1 ("late gene products") required at least three GGGCGG boxes, sequences between nucleotides 197 and 273 in the 72-bp repeat region, and transactivation by T-Ag. These results are consistent with the requirements for expression of the same genes in differentiated mammalian cells. Surprisingly, however, the 72-bp repeats ("enhancer elements") that are required for expression of T-Ag and t-Ag genes in differentiated cells were not required in mouse oocytes. Similarly, expression of both the early and late genes was unaffected in mouse oocytes by the absence of either DNA replication or an intact ori sequence, components required for maximum expression of late genes in differentiated cells. Thus, mammalian oocytes effectively utilize promoters that are fully active in mammalian differentiated cells only when associated with either enhancer elements or DNA replication. Furthermore, requirements for expression of SV40 genes in mouse oocytes are distinctly different from those reported for Xenopus oocytes. This suggests that caution should be exercised when extrapolating conclusions drawn from experiments with amphibian germ cells to mammalian germ cells.

Embryo infiltration by maternal macrophages is associated with selective expression of proto-oncogenes in a murine model of spontaneous abortion.

The causes and precise mechanisms leading to early embryo loss in mammals remain largely unknown, especially from a molecular point of view. Using the CBA/J x DBA/2 murine model of early spontaneous embryo loss (25-30% embryo loss), we have previously demonstrated the involvement of infiltrating activated macrophages and their cytolytic products such as nitric oxide and tumor necrosis factor alpha (TNF alpha) in the etiology of early embryo loss. On the other hand, far fewer of the CBA/J x Balb/c conceptuses (5-10% embryo loss) displayed significant cellular infiltration and nitric oxide and TNF alpha. Having used probes for cellular activation markers, we now present evidence indicating that significantly increased expression of AP-1 family members, Ha-ras, Ki-ras, v-erbA, v-raf, v-abl, and c-myc was present in 24.4% of the CBA/J x DBA/2 embryonic units that also harbored significant Mac-1, F4/80, and class II major histocompatibility complex (MHC) molecule cellular infiltration. In contrast, only 7% of CBA/J x Balb/c conceptuses displayed increased proto-oncogene expression and increased cellular infiltration. Therefore, macrophage infiltration, cellular activation as identified by the increased expression of proto-oncogenes, and the production of cytotoxic macrophage products are closely linked to early embryo loss. These data add to the evidence that activated maternal macrophages may be directly responsible for spontaneous pregnancy failure.