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P Celano

Publications and source records attributed to P Celano.

At least 19 recordsLinked to original sources
PubMed1992

Steady-state messenger RNA and activity correlates with sensitivity to N1,N12-bis(ethyl)spermine in human cell lines representing the major forms of lung cancer.

Our previous results from a limited number of cell lines have suggested that the bis(ethyl)polyamine analogues exert a phenotype-specific response in human lung cancer cells. In the present study, we have extended this work to analyze the response of the 4 major forms of human lung cancer to the polyamine analogue N1,N12-bis(ethyl)spermine (BESpm). The results suggest that non-small cell phenotypes are much more sensitive to the cytotoxic effects of BESpm than the small cell lung carcinoma phenotype. Further, there appears to be a positive association between the level of induction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT) in response to the analogue and the kinetic response of cells. Specifically, cells in which SSAT activity is highly induced by BESpm are killed by the compound. Although induction of SSAT appears to occur at both the level of increased steady-state mRNA and enzyme activity, SSAT activity appears to be a better indicator of cell sensitivity to BESpm than SSAT mRNA levels. These results have significance both for the potential use of polyamine analogues in treating specific forms of human lung cancer and for understanding the regulation of SSAT at the molecular level.

Acetyltransferases↗
PubMed1992

Structure of the human spermidine/spermine N1-acetyltransferase gene (exon/intron gene organization and localization to Xp22.1).

The super induction of spermidine/spermine N1-acetyltransferase (SSAT), has been implicated in the cytotoxic response of human solid tumors to the bis(ethyl)polyamines. The SSAT response is a phenotype specific response and is modulated at the level of increased steady-state mRNA levels and enzyme protein. The human genomic region (4,095 bases) containing the coding sequence of SSAT has been cloned and localized to the Xp22.1 region. Primer extension analysis indicates the transcription of SSAT starts 179 bases upstream from the translational start site and appears to be under the control of a "TATA-less" promoter. The availability of this human clone will facilitate the direct functional examination of the SSAT gene.

Acetyltransferases↗
PubMed1992

Characterization of an endogenous RNA transcript with homology to the antisense strand of the human c-myc gene.

In addition to being regulated by a complex array of cis- and trans-acting factors, c-myc protooncogene expression may be modulated by antisense RNA transcripts. Our previous studies have determined that depletion of intracellular polyamines by alpha-difluoromethylornithine results in a marked decrease in the transcription of the human c-myc gene. Because of reports that antisense transcription occurs in the 5' and 3' regions of this gene, we used a genomic clone of the human c-myc gene to ascertain whether polyamine depletion might induce an antisense RNA transcript. These studies demonstrate that polyamine depletion of the human colon cancer cell line COLO 320 results in induction of an endogenous RNA transcript with high homology to the antisense strand of the second intervening sequence (PvuII-RsaI) of the c-myc gene. Furthermore, during such depletion, steady state levels of this transcript vary inversely to the sense direction c-myc RNA. RNase protection studies suggest that the antisense transcript may arise from a different gene locus than the c-myc gene. To further identify the origins of this RNA, a cDNA library was generated from size-selected RNA and screened with c-myc sequences. A 438-base pair cDNA was isolated with approximately 85% homology, to a 285-base region in the second intron of the c-myc gene. Computer homology analysis further reveals that a 120-base region within this cDNA also has approximately 85% homology to the antisense strands of a number of genes, including the growth-related genes, N-myc, p53, and thymidine kinase. These studies provide the initial characterization of an endogenous antisense RNA transcript which could influence cell growth by modulating the expression of c-myc and other genes.

Base Sequence↗
PubMed1992

Isolation and characterization of the cDNA encoding human DNA methyltransferase.

We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase). This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa. The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells. Like the murine DNA MTase, the amino terminal two-thirds of the human protein contains a cysteine-rich region suggestive of a metal-binding domain. The carboxy terminal one-third of the protein shows considerable similarity to prokaryotic (cytosine-5)-methyltransferases. The arrangement of multiple motifs conserved in the prokaryotic genes is preserved in the human DNA MTase, including the relative position of a proline-cysteine dipeptide thought to be an essential catalytic site in all (cytosine-5)-methyltransferases. A single 5.2 kb transcript was detected in all human tissues tested, with the highest levels of expression observed in RNA from placenta, brain, heart and lung. DNA MTase cDNA clones were used to screen a chromosome 19 genomic cosmid library. The DNA MTase-positive cosmids which are estimated to span a genomic distance of 93 kb have been localized to 19p13.2-p13.3 by fluorescence in situ hybridization. Isolation of the cDNA for human DNA MTase will allow further study of the regulation of DNA MTase expression, and of the role of this enzyme in establishing DNA methylation patterns in both normal and neoplastic cells.

Amino Acid Sequence↗
PubMed1992

Cellular localization of retinoic acid receptor-gamma expression in normal and neoplastic skin.

Retinoids profoundly affect the normal growth and differentiation of epithelial tissues. Retinoic acid receptor-gamma (RAR-gamma) is a member of a family of retinoid receptors, and has been shown to be expressed almost exclusively in skin. However, little is known about the cellular localization of this receptor in human skin. The authors studied the expression of RAR-gamma in normal skin and human skin tumors by Northern blot analysis and in situ hybridization. RAR-gamma mRNA was detected in normal skin as well as in cultures of neonatal keratinocytes. Using an oligonucleotide specific for the RAR-gamma cDNA isoform 1 (RAR-gamma 1), RAR-gamma 1 mRNA was localized to all layers of the epidermis, the outer root sheath of hair follicles, follicular hair bulbs, eccrine and sebaceous glands. Basal cell carcinoma constitutively expressed gamma-1 mRNA and one of seven squamous cell carcinomas showed loss of gamma-1 mRNA expression, relative to adjacent epithelium. By contrast, normal melanocytic nevi and tumor-associated lymphocytes expressed little or no RAR-gamma mRNA. These results suggest that RAR-gamma 1 may play an important role in the maintenance and differentiation of normal epidermis and skin appendages.

Blotting, Northern↗
PubMed1991

Characterization of a full-length cDNA which codes for the human spermidine/spermine N1-acetyltransferase.

Spermidine/spermine N1-acetyltransferase is the rate-limiting enzyme in the catabolism of cellular polyamines. Using a combination of cDNA library screening and anchored PCR methodologies, a full length cDNA designated AP3/F7 corresponding to the human SSAT was cloned using RNA from the human large cell undifferentiated lung carcinoma line NCI H157. The resulting cDNA clone is 1,060 base pairs with a 513 base open reading frame coding for a 171 amino acid protein, with a predicted subunit molecular weight of 20,023. The 5' non-coding region of AP3/F7 is 165 bases and the 3' untranslated region is 382 bases with a polyadenylation site 20 bases 5' to the poly(A) tail. This full length cDNA should be an aid in the study of the regulation of spermidine/spermine N1-acetyltransferase expression and the significance of the acetyltransferase in polyamine metabolism.

Acetyltransferases↗
PubMed1991

High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer.

DNA methylation abnormalities occur consistently in human neoplasia including widespread hypomethylation and more recently recognized local increases in DNA methylation that hold potential for gene inactivation events. To study this imbalance further, we have cloned and localized to chromosome 19 a portion of the human DNA methyltransferase gene that codes for the enzyme catalyzing DNA methylation. Expression of this gene is low in normal human cells, significantly increased (30- to 50-fold by PCR analysis) in virally transformed cells, and strikingly elevated in human cancer cells (several hundredfold). In comparison to colon mucosa from patients without neoplasia, median levels of DNA methyltransferase transcripts are 15-fold increased in histologically normal mucosa from patients with cancers or the benign polyps that can precede cancers, 60-fold increased in the premalignant polyps, and greater than 200-fold increased in the cancers. Thus, increases in DNA methyltransferase gene expression precede development of colonic neoplasia and continue during progression of colonic neoplasms. These increases may play a role in the genetic instability of cancer and mark early events in cell transformation.

Animals↗
PubMed1991

Isolation and characterization of a cDNA clone that codes for human spermidine/spermine N1-acetyltransferase.

Spermidine/spermine N1-acetyltransferase (Spd/Spm acetyltransferase) is the rate-limiting enzyme in the catabolism of polyamines. This enzyme is highly inducible by several stimuli, including the natural polyamines and their structural analogues. To investigate the underlying mechanism responsible for the control of this enzyme a cDNA which codes for an active human Spd/Spm acetyltransferase has been isolated from a random primed cDNA library constructed from mRNA of a polyamine analogue treated large cell lung carcinoma line, NCI H157. The 972-base pair cDNA was identified using a 32-fold degenerate, 20-base oligomer probe to a 7-amino acid polypeptide sequence derived from the purified protein. The cDNA has a 513-base open reading frame that codes for a protein of 171 amino acids with a predicted molecular weight of 20,023. In vitro translation studies demonstrated the protein product of this cDNA to be a biologically active enzyme. The cDNA recognizes a 1.5-kilobase transcript in human cells which is highly induced in the human large cell lung carcinoma NCI H157 line following treatment with the polyamine analogue. The unusually high expression of Spd/Spm acetyltransferase mRNA by the NCI H157 cells in response to treatment does not appear to be a result of an amplification of the Spd/Spm acetyltransferase gene.

Acetyltransferases↗
PubMed1991

Colorectal neoplasia in juvenile polyposis or juvenile polyps.

Juvenile (retention) polyps are usually solitary lesions in the colorectum but may be multiple in juvenile polyposis. The association between juvenile polyps and colorectal neoplasia is controversial. We present three patients with juvenile polyposis who had colorectal adenomas or adenomatous epithelium in juvenile polyps at ages 3, 4, and 7 years. In a retrospective study of 57 additional patients with one or more juvenile polyps, 10 patients (18%) had colorectal neoplasia including three with adenocarcinoma, two with tubular adenoma, and six with adenomatous epithelium in a juvenile polyp (one had both adenomatous epithelium and an adenocarcinoma). Nine of these 10 patients had juvenile polyposis defined by the presence of at least three juvenile polyps; and eight of the nine had a family history of juvenile polyps. Colorectal neoplasia occurred at young age (mean (SEM) 37 (5) years). Our findings suggest that patients with juvenile polyps who have three or more juvenile polyps or a family history of juvenile polyps should undergo surveillance for colorectal neoplasia.

Adenocarcinoma↗
PubMed1990

High specific induction of spermidine/spermine N1-acetyltransferase in a human large cell lung carcinoma.

The cytotoxic response of the human large cell lung carcinoma line NCI H157 to exposure to the polyamine analogue N1 N12-bis(ethyl)spermine (BESpm) is preceded by an extremely high induction of spermidine/spermine N1-acetyltransferase (SSAT). The human enzyme has now been purified greater than 300-fold to apparent homogeneity and found to cross-react with antisera raised against rat liver SSAT. Although other acetylases are capable of acetylating polyamines using acetyl-CoA as the acetyl donor, the greater than 600-fold induction within 24 h was found to be specifically SSAT, since essentially all activity was precipitable by the specific antisera. The human enzyme appears to be similar to the rat enzyme in subunit size under reducing conditions (approximately 20 kDa), substrate specificity and kinetic parameters. Preliminary results using actinomycin D and cycloheximide suggested that the unusually high induction by N1 N12-bis(ethyl)spermine in the human lung cancer line result from new mRNA and protein synthesis. This hypothesis is further substantiated here by 'in vitro' translation experiments comparing poly(A) mRNA from control and treated cells. The large cell lung carcinoma line NCI H157 represents a useful system to produce large amounts of the SSAT protein and to study the molecular events responsible for the induction and control of this important polyamine-metabolic enzyme. By using this rich source of SSAT protein, a partial amino acid sequence was determined by N-terminal sequencing of endoproteinase Lys-C digestion fragments. Further, this system should be useful in determining whether there is an association between the unusually high induction of the acetylase and the observed cytotoxicity in the NCI H157 cells.

Acetyltransferases↗
PubMed1990

Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells.

Long-term reconstitution of the lymphohaematopoietic cells of a mouse after lethal irradiation requires the transplantation of at least (5-10) x 10(3) bone marrow cells. Several cell-separation techniques based on cell-surface characteristics have been used in attempts to identify the pluripotent haematopoietic stem cells (PHSC), and have allowed the long-term engraftment of lethally irradiated mice with an enriched fraction of fewer than 200 marrow cells. But these techniques enrich not only for PHSC but also for haematopoietic progenitors, especially day-12 spleen colony-forming units (CFU-S). Although day-12 CFU-S have been postulated to be primitive multipotential haematopoietic progenitors, with day-8 CFU-S representing later, more committed progenitors, recent evidence suggests that neither of these CFU-S represents mouse PHSC. Here we report that counterflow centrifugal elutriation, which sorts cells on the basis of size and density, can separate PHSC from these less primitive progenitors. The fraction containing the largest cells was enriched for the granulocyte-macrophage colony-forming units (CFU-GM), but gave only transient, early engraftment and was therefore depleted of PHSC. The intermediate fraction was enriched for CFU-S, but depleted of CFU-GM. Despite being devoid of CFU-GM and CFU-S, the fraction consisting of only morphological lymphocytes gave sustained, albeit delayed, reconstitution of all lymphohaematopoietic cells, and was therefore enriched for PHSC. We conclude that there are two vital classes of engrafting cells: committed progenitors, which provide initial, unsustained engraftment, and PHSC, which produce delayed, but durable, engraftment. Therefore for late haematological reconstitution, PHSC must be transplanted with a distinguishable source of early engrafting cells, thereby allowing the lethally irradiated host to survive initial aplasia.

Animals↗
PubMed1989

Modulation of growth gene expression by selective alteration of polyamines in human colon carcinoma cells.

The biosynthesis of the polyamines, putrescine, spermidine and spermine, is temporally linked with expression of many growth related genes. Our previous studies have shown that generalized polyamine depletion of the human colon cancer cell line COLO 320 by 2-difluoromethylornithine is associated with decreased transcription of the c-myc, c-fos, and ornithine decarboxylase (ODC) genes. In the current study, the role of individual polyamines was further defined by the use of a specific inhibitor of spermidine synthase, S-adenosyl-1,8, diamino-3-thio-octane (AdoDATO), and a spermine analogue, N1,N12 bis(ethyl)spermine. Our data demonstrate that depletion of spermidine results in a 60-90% decrease in c-myc mRNA steady state levels. In contrast, c-fos mRNA levels are decreased only when both spermidine and spermine are diminished. Furthermore, ODC mRNA levels are increased when all polyamines are decreased by DFMO, but are unaffected by a selective reduction in intracellular spermidine levels by AdoDATO. These studies suggest that individual polyamines may have a selective role in the expression of specific growth related genes.

Adenosine↗
PubMed1989

Differential induction of spermidine/spermine N1-acetyltransferase in human lung cancer cells by the bis(ethyl)polyamine analogues.

We have investigated the induction of an important polyamine metabolic enzyme, spermidine/spermine N1-acetyltransferase, in two human lung cancer cell lines which respond differently to treatment with the bis(ethyl)polyamine analogues. The human small cell lung carcinoma line NCI H82 has previously been shown to be minimally affected by treatment with these analogues, whereas the large cell undifferentiated lung carcinoma line, NCI H157, responds in a rapid cytotoxic manner (R.A. Casero, Jr., S. J. Ervin, P. Celano, S. B. Baylin, and R. J. Bergeron, Cancer Res., 49:639-643, 1989). The mechanisms underlying the differential response are unknown. In the responsive NCI H157 cells, the bis(ethyl)polyamines were found to induce spermidine/spermine N1-acetyltransferase in a time- and dose-dependent manner to maximum levels greater than 1700-fold over baseline. By contrast, the unresponsive NCI H82 cells exhibit minimal induction of spermidine/spermine N1-acetyltransferase to less than 7-fold increase after bis(ethyl)polyamine treatment, regardless of time or concentration examined. The results of the current study suggest that the differential induction of this key enzyme, which is rate limiting in the back conversion pathway of polyamine metabolism, may play a role in determining cell specific to the bis(ethyl)polyamine analogues.

Acetyltransferases↗
PubMed1989

Polyamines differentially modulate the transcription of growth-associated genes in human colon carcinoma cells.

Increases in the transcription of genes important to cell growth frequently occur in concert with increases in intracellular polyamines after a mitogenic stimulus. Previously, we have shown that depletion of intracellular polyamines by 2-difluoromethylornithine in COLO 320 cells resulted in a significant decrease in c-myc mRNA steady state levels. We demonstrate here that polyamines have a predominant role in the regulation of transcription of c-myc, c-fos, and histone 2A, while they appear to have both a transcriptional and post-transcriptional role in expression of c-myb, ornithine decarboxylase, and beta-actin mRNA levels. We further analyzed the effect of spermidine in overall and specific RNA transcription in isolated nuclei from COLO 320 cells. In nuclei from control cells, the addition of spermidine resulted in a 3-4 increase in overall [alpha-32P]UTP incorporation and a 4-8-fold increase in c-myc, c-fos, and histone 2A transcription. In contrast, ornithine decarboxylase and c-myb gene transcription were unaffected. Furthermore, in nuclei from 2-difluoromethylornithine-treated COLO 320 cells, spermidine addition resulted in less than a 2-fold increase in RNA transcription and had no effect on any specific gene analyzed. These results indicate that polyamines may have an important role in the transcription of specific growth related genes, especially c-myc and c-fos, and this role may be one mechanism by which polyamines modulate cell growth.

Actins↗
PubMed1989

Differential response to treatment with the bis(ethyl)polyamine analogues between human small cell lung carcinoma and undifferentiated large cell lung carcinoma in culture.

We have compared the effects of treatment with each of three bis(ethyl)polyamine analogues on a human small cell lung carcinoma (SCLC) line, NCI H82, and a non-small cell line, NCI H157, an undifferentiated large cell lung carcinoma. The bis(ethyl)polyamines have been shown to interfere with polyamine metabolism, presumably by regulation of the polyamine biosynthetic pathway in a manner similar to the natural polyamines, in contrast to direct inhibition of specific enzymes, such as ornithine decarboxylase. Each of these compounds was found to be relatively inactive in reducing growth rate, polyamine levels, or polyamine biosynthetic enzyme activity in the SCLC cells, a line which we have previously shown to be particularly sensitive to inhibition of polyamine biosynthesis by the direct ornithine decarboxylase inhibitor difluoromethylornithine. By contrast, each of the bis(ethyl)polyamines tested was found to be markedly cytotoxic (at concentrations of only 10 microM) to the non-SCLC line, NCI H157. Interestingly, the non-SCLC line has previously been demonstrated to be resistant to polyamine depletion by difluoromethylornithine. For each bis(ethyl)polyamine, cytotoxicity was accompanied by nearly complete depletion of all intracellular polyamines and a decrease in ornithine decarboxylase activity to undetectable levels. The current study emphasizes the phenotypic variability which can exist in response to inhibitors of polyamine biosynthesis and suggests a class of agents which may have clinical utility against the treatment-resistant non-SCLC lung cancers.

Adenosylmethionine Decarboxylase↗
PubMed1989

Two phases of engraftment established by serial bone marrow transplantation in mice.

Serially transplanted bone marrow eventually fails to reconstitute lethally irradiated mice. The reasons for this loss of repopulating ability are unknown. We showed that serial bone marrow transplantation changed the ratio of hematopoietic progenitors in bone marrow. The numbers of granulocyte-macrophage colony-forming units (CFU-GM) in the bone marrow did not change with serial transplantation. Spleen CFU (CFU-S) numbers decreased with serial transfer but remained at levels which should be associated with engraftment, even on the transfers which were unsuccessful. The CFU-S, therefore, did not appear to be the cells responsible for long-term hematopoietic repopulation. The number of successful serial transfers was dependent on the size of the grafts, and prolonging the time interval between transfers reestablished the ability of the serially transplanted marrow to reconstitute lethally irradiated recipients. Serial bone marrow transplantation dissociated two phases of engraftment. The first unsustained phase was maintained with repeated serial transfer and appeared to be produced by committed progenitors. The second sustained phase was eventually lost with repeated serial transfer and was apparently due to the pluripotent stem cell.

Animals↗
PubMed1988

Effect of polyamine depletion on c-myc expression in human colon carcinoma cells.

Polyamine depletion generally results in an inhibition of cell growth. However, the precise role of polyamines in the regulation of cell proliferation is unknown. In the present study, we demonstrate that polyamine depletion induced by 2-difluoromethylornithine in COLO 320 human colon carcinoma cells results in a greater than 90% decrease in expression of a key gene in the maintenance of cell growth, the c-myc protooncogene. The decrease in c-myc expression accompanying polyamine depletion appears to occur at the transcriptional level. It is not simply a result of decreased growth rate since growth-inhibited cells at confluence maintain a high level of c-myc expression. It is also due to a change in cell cycle phase distribution and is not a reflection of a generalized decrease in gene expression which accompanies a decrease in cellular proliferation. Thus, the expression of the histone H2A gene was similar to and temporally paralleled the growth status of both treated and untreated cells, while the beta-actin and ornithine decarboxylase genes actually had increased expression during polyamine depletion. These studies demonstrate that polyamines may be critical to the expression of c-myc and suggest one mechanism by which they modulate cell growth.

Actins↗