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M Caruso

Publications and source records attributed to M Caruso.

At least 73 records · Page 4Linked to original sources
PubMed1996

Characterization of two novel YY1 binding sites in the polyomavirus late promoter.

NF-D is a ubiquitous nuclear factor that has been shown to bind specifically to a DNA element in the polyomavirus regulatory region. In this report, we demonstrate that NF-D is either identical or very similar to a transcription factor that has been variously named YY1, delta, NF-E1, UCRBP, or CF1. Moreover, we show the presence in the polyomavirus genome of a second DNA motif, located 40 bp from the first, which binds YY1/NF-D with high affinity. Both sites lie downstream of the major late transcription initiation sites. By site-directed mutagenesis, we demonstrate that both elements contribute positively to the activity of the late promoter, probably by a cooperative mechanism. We also demonstrate that the requirement of the YY1/NF-D function for late promoter activity varies with the cell line.

3T3 Cells↗
PubMed1995

Decreased phosphorylation of mutant insulin receptor by protein kinase C and protein kinase A.

We have recently reported that the Arg1152-->Gln insulin receptor mutation (QK single mutant) alters a conserved motif (RK motif) immediately next to the key tyrosine phosphorylation sites (Tyr1146, Tyr1150, Tyr1151) of the receptor and constitutively activates its kinase and metabolic signaling. To investigate further the function of the RK motif, we have expressed two additional mutant insulin receptors: a single mutant, in which the second basic residue in the RK motif (Lys1153) was substituted (RA mutant); and a double mutant, in which both the Arg and the Lys residues were replaced with noncharged amino acids (QA mutant). As compared with the transfected wild-type receptors (WT), both the single and the double mutant receptors were normally synthetized and transported to the plasma membrane and bound insulin normally. Whereas the double mutant receptor exhibited preserved insulin-dependent autophosphorylation, kinase activity, and 2-deoxyglucose uptake, all of these functions were grossly impaired in the two single mutant receptors. Two-dimensional analysis of tryptic phosphopeptides from receptor beta-subunits revealed that decreased autophosphorylation of the single mutant receptors mainly involved regulatory Tyr1150,1151 and carboxyl-terminal Tyr1316,1322. At variance with the insulin-stimulated, insulin-independent tyrosine kinase activity toward poly(Glu-Tyr) 4:1 was increased 3-fold in both the double and the single mutants. All mutant receptors induced a 2-fold increase in basal 2-deoxyglucose uptake in NIH-3T3 cells. Treatment of WT transfected cells with 12-O-tetradecanoyl-phorbol-13-acetate or 8-bromo-cAMP increased insulin receptor phosphorylation by 3-fold. No phosphorylation was observed in cells expressing the two single or the double mutant receptor. Consistently, purified preparations of PKC and PKA phosphorylated the WT but not the mutant receptors in vitro. A 17-amino acid synthetic peptide encoding the receptor sequence surrounding the RK motif inhibited phosphorylation of WT insulin receptors by both protein kinases A and C. A mutant peptide in which the RK sequence was replaced by QK (to mimic the mutation in the QK receptor) exhibited no inhibitory effect. Thus, the RK insulin receptor motif is required for insulin receptor phosphorylation by protein kinases C and A and may modulate insulin-independent receptor activity. The RK motif may also have an important structural role in allowing normal insulin regulation of the kinase.

3T3 Cells↗
PubMed1995

Expression of a Tat-inducible herpes simplex virus-thymidine kinase gene protects acyclovir-treated CD4 cells from HIV-1 spread by conditional suicide and inhibition of reverse transcription.

Cellular expression of the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene promotes cell death in the presence of specific nucleoside analog substrates such as acyclovir (ACV). We have reported that lymphoid CD4+ cells harboring an HSV1-TK gene, under the transcriptional control of the HIV-1 long terminal repeat (HUT-TK), are completely protected from HIV-1 spread in the presence of 10 microM ACV. In this report we clarify the efficiency, generality, and mechanism of this protective effect. We show that the protection from HIV-1 spread in HUT-TK cells obtains from both an inhibition of HIV reverse transcription by ACV metabolites and an HIV-induced and ACV-dependent cell killing. We also demonstrate that monocytic cells harboring the HIV-1-inducible HSV1-TK gene are protected from HIV spread in the presence of ACV. These observations facilitate the design of therapeutic strategies to limit HIV replication based on HSV1-TK expression.

Acyclovir↗
PubMed1995

The inhibition of cultured myoblast differentiation by the simian virus 40 large T antigen occurs after myogenin expression and Rb up-regulation and is not exerted by transformation-competent cytoplasmic mutants.

We have investigated the mechanism by which the simian virus 40 large T antigen (SVLT) interferes with the differentiation of C2 myoblasts. SVLT mutants, defective either in the Rb binding site, near the N-terminal end, in a region that affects binding to p53, or in the nuclear transport signal, were also employed to determine whether the interference was especially dependent on these functional domains. It was found that wild-type (wt) SVLT strongly inhibited the terminal differentiation of mouse C2 myoblasts, but this arrest occurred only after the synthesis of myogenin, an initial step in biochemical differentiation. Neither the synthesis nor some basic activities of MyoD appeared to be affected by wt SVLT. In these transformants, mitogen depletion elicited an increase in the Rb level comparable to that in normal C2 cells; wt SVLT, however, promoted the phosphorylation of a large part of the induced Rb. Mutations affecting nuclear transport were far more critical for the ability to interfere with myogenic differentiation than were those affecting the transforming potential; cytoplasmic SVLT expression was fully compatible with the terminal differentiation of C2 cells, despite enabling them to grow in semisolid medium, thus showing that the myogenesis-inhibiting property can be dissociated from transforming competence. The remaining SVLT mutants presented different degrees of ability to inhibit differentiation (as shown by the expression of tissue-specific markers in transformants). The inhibiting mutants, including the Rb binding site mutant, were able to promote a higher state of Rb phosphorylation than that observed in either normal cells or cytoplasmic-SVLT transformants.

Actins↗
PubMed1995

A truncated herpes simplex virus thymidine kinase phosphorylates thymidine and nucleoside analogs and does not cause sterility in transgenic mice.

Dividing eukaryotic cells expressing the herpes simplex virus type 1 thymidine kinase (TK) gene are sensitive to the cytotoxic effect of nucleoside analogs such as acyclovir or ganciclovir (GCV). Transgenic mice with cell-targeted expression of this conditional toxin have been used to create animals with temporally controlled cell-specific ablation. In these animal models, which allow the study of the physiological importance of a cell type, males are sterile. In this study, we showed that this phenomenon is due to testis-specific high-level expression of short TK transcripts initiated mainly upstream of the second internal ATG of the TK gene. This expression is DNA methylation independent. To obtain a suicide gene that does not cause male infertility, we generated and analyzed the properties of a truncated TK (delta TK) lacking the sequences upstream of the second ATG. We showed that when expressed at sufficient levels, the functional properties of delta TK are similar to those of TK in terms of thymidine or GCV phosphorylation. This translated into a similar GCV-dependent toxicity for delta TK- or TK-expressing cells, both in vitro and in transgenic mice. However, delta TK behaved differently from TK in two ways. First, it did not cause sterility in delta TK transgenic males. Second, low-level delta TK RNA expression did not confer sensitivity to GCV. The uses of delta TK in cell-specific ablation in transgenic mice and in gene therapy are discussed.

Animals↗
PubMed1995

Sequence-specific DNA interactions by novel alkylating anthracycline derivatives.

New alkylating anthracycline derivatives with promising antitumor activity have been synthesized. We selected two of these compounds, 4-demethoxy-N,N-bis(2 chloroethyl)-4'-methylsulfonyl-daunorubicin (FCE 27726) and 4-demethoxy-3'-deamino-3'aziridinyl-4'-methylsulfonyl daunorubicin (FCE 28729), comparing their interaction with DNA and that of the non-alkylating derivative 4-demethoxy-4'-methylsulfonyl-daunorubicin (FCE 27894). The two alkylating derivatives were more cytotoxic than idarubicin and presented low cross-resistance with doxorubicin. Both FCE 27726 and FCE 28729 were found to alkylate guanines at the N7 position in the major groove with roughly the same specificity, but at different concentrations. FCE 27726 was 10 times more potent than FCE 28729 in alkylating DNA. At higher concentrations, FCE 27726 was able to alkylate adenines, possibly at the N3 position contained in a sequence 5'-PyAA. FCE 27726, as expected, was able to form DNA interstrand cross-links either in vitro and in vivo in treated cells. FCE 28729 did not form DNA interstrand cross-links in vivo. In vitro, at high concentrations, some DNA interstrand cross-links were evident. The non-alkylating derivative FCE 27894 did not produce any alkylation or DNA interstrand cross-links either in vitro or in vivo.

Animals↗
PubMed1994

Internalization of the constitutively active arginine 1152-->glutamine insulin receptor occurs independently of insulin at an accelerated rate.

Signals controlling the insulin receptor endocytotic pathway have been investigated using the R1152Q insulin receptor mutant (M). This mutant receptor exhibits high levels of insulin-independent kinase activity, impaired autophosphorylation, and lack of an insulin stimulatory effect on both auto- and substrate phosphorylation. NIH-3T3 fibroblasts expressing M receptors displayed a 2.5-fold higher 125I-insulin internalization rate than wild type (WT) but lacked insulin-induced receptor internalization and down-regulation. Cell surface recycling of internalized receptors also occurred at a higher rate in M cells and was unaffected by insulin. Cell preincubation with 35 mM Tris, which inhibits the insulin receptor degradative route, elicited no effect on M receptor recycling but inhibited that of WT by 40%. In contrast, the energy depleter 2,4-dinitrophenol, which inhibits normal insulin receptor retroendocytosis, impaired M receptor recycling 4-fold more effectively than that of WT. The release of internalized intact 125I-insulin was 6-fold greater in M than in WT fibroblasts and was almost completely inhibited by dinitrophenol, whereas insulin degradation by M cells was 4-fold decreased as compared with WT. Thus, internalization and recycling of the constitutively active Gln1152 receptor kinase occur in the absence of autophosphorylation. However, tyrosine phosphorylation appears to be required for proper sorting of endocytosed insulin receptors.

2,4-Dinitrophenol↗
PubMed1994

Primary head and neck cancer. Histopathologic predictors of recurrence after neck dissection in patients with lymph node involvement.

OBJECTIVE: Retrospectively analyze several histopathologic variables that may predict neck recurrence after neck dissection. DESIGN: From 1970 through 1980, 284 patients with pathologically confirmed metastatic squamous cell carcinoma underwent neck dissection and received no adjuvant therapy. Kaplan-Meier evaluation estimated a 74% 2-year neck recurrence-free rate. After adjusting for the standard covariates of age, gender, neck stage, and tumor grade, we also controlled for the time-dependent covariates of primary recurrence, occurrence in the side of the neck not operated on, or development of new head and neck primary disease. SETTING: A large referral-based practice. RESULTS: The number of lymph nodes involved, invasion of vascular/lymphatic space, invasion of soft tissue, and desmoplastic lymph node pattern adversely affect neck recurrence. A desmoplastic stromal pattern was associated with almost a sevenfold increased risk of neck recurrence. To our knowledge, this finding has not been reported previously. CONCLUSION: Histopathologic evaluation of metastatically involved cervical nodes can identify patients with head and neck cancer who are at high risk for recurrence.

Adult↗
PubMed1994

Development of clinical standards in industrial rehabilitation.

Before discussing adjustments in the fees for the clinical services of industrial rehabilitation, the Medical Free Guide Committee of the Maryland Industrial Commission requested a copy of the clinical standards for these services. However, there were no multidisciplinary standards for industrial services that had state-wide approval. Therefore, a committee was formed to write the standards. Following a review of the literature and unpublished work from other associations and state organizations, standards were developed. Constructive criticism was solicited from national and local professionals and organizations with demonstrated interest and experience in providing or using these services or with experience writing standards. Further comment was solicited from individuals in the state of Maryland before final editing. Because of the dearth of outcome studies supporting the efficacy of clinical services in industrial rehabilitation, the standards describing a level of expectation from clinical services have become important for state organizations responsible for making decisions on reimbursement for clinical services. This paper summarizes the process used for the development of clinical standards of industrial rehabilitation services.

Contraindications↗
PubMed1994

[Genetically controlled pharmacomodulation: application to the treatment of HIV infection].

AZT is an inhibitor of HIV reverse transcriptase which active form is AZT-triphosphate. Its clinical efficacy is limited by its toxicity and the emergence of mutant resistant viruses. This might be due to an unfficient cellular metabolism of AZT which leads to the intracellular accumulation of AZT monophosphate. We tested a possible enhancement of this metabolism with the HSV1 thymidine kinase, an enzyme that also possesses a good thymidilate kinase activity. We show that, compared to parental cells, the proportion of AZT triphosphate is increased 3 fold in HSV1-TK expressing cells, and that inhibition of HIV replication in these cells requires 3 to 10 fold less AZT. This observation forms the basis for a new gene therapy strategy named genetically controlled pharmacomodulation.

Cell Line, Transformed↗
PubMed1994

MyoD induces retinoblastoma gene expression during myogenic differentiation.

In skeletal muscle cells permanent withdrawal from the cell cycle is a prerequisite for terminal differentiation. The muscle-specific transcription factor MyoD can activate downstream muscle structural genes and myogenic conversion in many different cell types. It has been demonstrated that the product of the retinoblastoma susceptibility gene, with its growth-suppressive activity, is involved in the myogenic function of MyoD (Caruso et al., 1993; Gu et al., 1993). The present study characterises the modulation of retinoblastoma (Rb1) mRNA levels during myogenic differentiation of the murine C2 cell line and provides evidence that the muscle-specific regulatory factor MyoD enhances Rb1 gene transcription. We demonstrate that MyoD mediates the transactivation of a CAT construct whose expression is driven by the human Rb1 gene promoter, and that this is not a consequence of direct binding of MyoD to an E-box DNA sequence motif present in the Rb1 promoter sequences. In addition we have tested the capability of several MyoD mutant proteins of inducing the Rb1 promoter CAT construct. Our results indicate that the MyoD function required for induction of Rb1 promoter activity is distinct from its myogenic function.

Animals↗
PubMed1993

Regression of established macroscopic liver metastases after in situ transduction of a suicide gene.

The herpes simplex virus type 1 thymidine kinase (HSV1-TK) converts nontoxic nucleoside analogs such as ganciclovir into phosphorylated compounds that act as chain terminators and specifically kill dividing cells. This property could be exploited for the treatment of tumors that are made up of rapidly dividing cells invading a nonproliferating tissue. For this purpose, specific expression of the suicide gene into dividing tumor cells can be further targeted by using retroviral-mediated gene transfer. We investigated whether the direct intratumoral transduction of a suicide gene might induce the elimination of malignant solid tumors. Rats with established macroscopic liver metastases were given an intratumoral injection of packaging cells producing either HSV1-TK- or lacZ-expressing recombinant retroviral particles. All rats were next treated with ganciclovir. A dramatic regression of the tumor volume was observed in the HSV1-TK-treated animals. The residual tumors were mostly made up of a massive fibrotic reaction, with the mean cancer cell mass being reduced approximately 60-fold compared to controls. In some animals, the residual tumors were devoid of cancer cells. This treatment efficacy appears in part due to a "bystander effect" in which phosphorylated ganciclovir could be transferred from cell to cell and to an active local immune reaction evidenced by massive infiltration of the tumors by macrophages and both CD4+ and CD8+ lymphocytes. This efficient therapeutic approach might be an ultimate treatment for disseminated liver metastases in humans and could also be applied to treatment of a large variety of solid tumors.

Animals↗
PubMed1993

Results of surgical treatment for growth hormone-secreting pituitary adenomas.

The results of surgical therapy for acromegaly were reviewed in a series of 175 patients treated between 1972 and 1983. Patients with prior surgery or radiation therapy were excluded from the study. Postoperative radiation therapy was given to 54 patients. The criterion of achieving a postoperative basal or glucose-suppressed growth hormone level of 2 ng/ml or less was used to indicate remission. Utilizing the most recently available growth hormone determinations, 90 (51.7%) of 174 patients were in remission. The actuarial probability of remission at 1 and 5 years after surgery was 48.8% and 62.7%, respectively. Tumor size and the preoperative basal growth hormone level were correlated with outcome. Surgical excision of a pituitary adenoma is the most effective therapy currently available for acromegaly.

Adenoma↗
PubMed1993

Chlamydia trachomatis diagnosis: a correlative study of pap smear and direct immunofluorescence.

From June 1989 to September 1990, 255 women with recurrent vaginitis, were evaluated, at the "Service of Precocious Diagnosis and Therapy of Gynecological Tumor" of the 1st Department of Obstetrics and Gynecology. They were compared with a control group of 255 women selected in the same time. All patients between the ages of 18-40 years, with normal sexual behaviour, had a negative colposcopy for HPV and HSV2 infections. The cervical smear with Papanicolaou technique and direct immunofluorescence (DIF) was performed in all patients. The specimens were examined with a fluorescence microscope (Leitz). Positive determinations were confined by examination a tha magnification of 600 X. Slides were scored as positive if at least 5 distinct apple-green fluorescence-stained elementary body per field was observed. The two groups were entirely comparable in age, parity, method of contraception, and number of sexual contacts over the preceding three months. Our data confirmed a higher incidence of Chlamydial infections in women with recurrent vaginitis (34.1%) than in control group (8.23%). In symptomatic women, more cases of metaplastic cells with cytoplasmic vacuolation, less inflammatory alteration and a lack of specific agents like CA and TV, were found than in the control group. The DIF positivity, in both groups, was connected with a cytological findings of metaplastic cells with cytoplasmic vacuolation in 72.3% and 50% respectively.

Adult↗
PubMed1993

Regulation of MyoD gene transcription and protein function by the transforming domains of the adenovirus E1A oncoprotein.

It has been demonstrated that the adenovirus E1A gene products inhibit myogenic differentiation in the mouse C2 muscle cell line. During myogenic differentiation, cell growth and tissue-specific gene expression are mutually exclusive. Since E1A exerts multiple effects on different cellular pathways through alteration of cell growth control and transcriptional regulation, we investigated in more detail the molecular mechanisms underlying the inhibitory effect of E1A on myogenic differentiation. To this end, we used mutant derivatives of E1A that lack the 'conserved domain' sequences to which the functional domains of E1A have been mapped, and we observed the effect of constitutive expression of these E1A mutants on myogenesis in the murine C2 muscle cell line. Our results demonstrate that E1A interferes with myogenesis through at least two mechanisms: (i) the inhibition of MyoD expression; (ii) the repression of MyoD-dependent transcriptional activation. In addition, we demonstrate also that the repression of MyoD transcription depends upon sequences located in the N-terminus of E1A and correlates well with the site of E1A/p300 association. Further, the inhibition of transcriptional activation by MyoD depends both on conserved region 1 and on conserved region 2, the two transforming domains of E1A. We demonstrate also that a similar inhibitory effect on the MyoD transactivating function is provided by the polyomavirus and SV40 large T oncoproteins.

Adenovirus E1A Proteins↗
PubMed1992

Selective killing of CD4+ cells harboring a human immunodeficiency virus-inducible suicide gene prevents viral spread in an infected cell population.

We have stably expressed in CD4+ lymphoid cells the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene under the control of the human immunodeficiency virus (HIV) promoter and transactivation response element sequences. Upon HIV infection these regulatory sequences were transactivated, switching on high-level expression of HSV1-TK. This in turn caused the death of HIV-infected cells when they were cultured in the presence of acyclovir, a nucleoside analog that becomes toxic after phosphorylation by HSV1-TK. The elimination of HIV-infected cells resulted in the arrest of HIV spreading in the culture. Complete protection of HSV1-TK-expressing cells was obtained using acyclovir concentrations that are commonly detected in the plasma of patients treated for HSV1 infection. Thus, expression of this DNA construct generates a pool of CD4+ booby-trapped cells that, as a population, are resistant to HIV infection. Our data provide a rationale for the use of suicide genes in the design of gene therapy of HIV infection.

Acyclovir↗