TGFß engages MEK/ERK to differentially regulate benign and malignant pancreas cell function.

While TGFß signals are anti-proliferative in benign and well-differentiated pancreatic cells, TGFß appears to promote the progression of advanced cancers. To better understand dysregulation of the TGFß pathway, we first generated mouse models of neoplastic disease with TGFß receptor deficiencies. These models displayed reduced levels of pERK irrespective of KRAS mutation. Furthermore, exogenous TGFß led to rapid and sustained TGFBR1-dependent ERK phosphorylation in benign pancreatic duct cells. Similar to results that our group has published in colon cancer cells, inhibition of ERK phosphorylation in duct cells mitigated TGFß-induced upregulation of growth suppressive pSMAD2 and p21, prevented downregulation of the pro-growth signal CDK2 and ablated TGFß-induced EMT. These observations suggest that ERK is a key factor in growth suppressive TGFß signals, yet may also contribute to detrimental TGFß signaling such as EMT. In neoplastic PanIN cells, pERK was not necessary for either TGFß-induced pSMAD2 phosphorylation or CDK2 repression, but was required for upregulation of p21 and EMT indicating a partial divergence between TGFß and MEK/ERK in early carcinogenesis. In cancer cells, pERK had no effect on TGFß-induced upregulation of pSMAD2 and p21, suggesting the two pathways have completely diverged with respect to the cell cycle. Furthermore, inhibition of pERK both reduced levels of CDK2 and prevented EMT independent of exogenous TGFß, consistent with most observations identifying pERK as a tumor promoter. Combined, these data suggest that during carcinogenesis pERK initially facilitates and later antagonizes TGFß-mediated cell cycle arrest, yet remains critical for the pathological, EMT-inducing arm of TGFß signaling.

Collagen regulation of let-7 in pancreatic cancer involves TGF-ß1-mediated membrane type 1-matrix metalloproteinase expression.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a pronounced collagen-rich fibrosis known as desmoplastic reaction; however, the role of fibrosis in PDAC is poorly understood. In this report we show that collagen can regulate the tumor suppressive let-7 family of microRNAs in pancreatic cancer cells. PDAC cells growing in 3D collagen gels repress mature let-7 without affecting the precursor form of let-7 in part through increased expression of membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) and ERK1/2 activation. PDAC cells in collagen also demonstrate increased TGF-ß1 signaling, and blocking TGF-ß1 signaling attenuated collagen-induced MT1-MMP expression, ERK1/2 activation and repression of let-7 levels. Although MT1-MMP overexpression was not sufficient to inhibit let-7 on 2D tissue culture plastic, overexpression of MT1-MMP in PDAC cells embedded in 3D collagen gels or grown in vivo repressed let-7 levels. Importantly, MT1-MMP expression significantly correlated with decreased levels of let-7 in human PDAC tumor specimens. Overall, our study emphasizes the interplay between the key proteinase MT1-MMP and its substrate type I collagen in modulating microRNA expression, and identifies an additional mechanism by which fibrosis may contribute to PDAC progression.

tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1.

Recent studies suggest a close relationship between cell metabolism and apoptosis. We have evaluated changes in lipid metabolism on permeabilized hepatocytes treated with truncated Bid (tBid) in the presence of caspase inhibitors and exogenous cytochrome c. The measurement of beta-oxidation flux by labeled palmitate demonstrates that tBid inhibits beta-oxidation, thereby resulting in the accumulation of palmitoyl-coenzyme A (CoA) and depletion of acetyl-carnitine and acylcarnitines, which is pathognomonic for inhibition of carnitine palmitoyltransferase-1 (CPT-1). We also show that tBid decreases CPT-1 activity by a mechanism independent of both malonyl-CoA, the key inhibitory molecule of CPT-1, and Bak and/or Bax, but dependent on cardiolipin decrease. Overexpression of Bcl-2, which is able to interact with CPT-1, counteracts the effects exerted by tBid on beta-oxidation. The unexpected role of tBid in the regulation of lipid beta-oxidation suggests a model in which tBid-induced metabolic decline leads to the accumulation of toxic lipid metabolites such as palmitoyl-CoA, which might become participants in the apoptotic pathway.

Highly invasive transitional cell carcinoma of the bladder in a simian virus 40 T-antigen transgenic mouse model.

Transitional cell carcinoma (TCC), a neoplasm of urinary bladder urothelial cells, generally appears in either of two forms, papillary non-invasive or invasive TCC, although intermediate forms can occur. Each has a distinctive morphology and clinical course. Altered expression of the p53 and pRb genes has been associated with the more serious invasive TCC, suggesting that the loss of activity of these tumor suppressor proteins may have a causal role in this disease. To test this hypothesis directly, transgenic mice were developed that expressed the simian virus 40 large T antigen (TAg) in urothelial cells under the control of the cytokeratin 19 gene (CK19) regulatory elements. In one CK19-TAg lineage, all transgenic mice developed highly invasive bladder neoplasms that resembled invasive human bladder TCCs. Stages of disease progression included development of carcinoma in situ, stromal invasion, muscle invasion, rapid growth, and, in 20% of affected mice, intravascular lung metastasis. Papillary lesions never were observed. Western blot analysis indicated that TAg was bound to both p53 and pRb, which has been shown to cause inactivation of these proteins. Our findings support suggestions that (i) inactivation of p53 and/or pRb constitutes a causal step in the etiology of invasive TCC, (ii) papillary and invasive TCC may have different molecular causes, and (iii) carcinoma in situ can represent an early stage in the progression to invasive TCC.

Kelletinin A, from the marine mollusc Buccinulum corneum, promotes differentiation in Hydra vulgaris.

The effects of kelletinin A [ribityl pentakis (p-hydroxybenzoate)] (KA), a natural compound isolated from the marine gastropod Buccinulum corneum, were studied in vivo in Hydra vulgaris during regeneration. KA caused a marked increase of regenerated tentacle numbers (ATN) and promoted transdifferentiation of epithelial cells into battery cells, and nematocyte differentiation. Morphological data were correlated to changes in acid and alkaline phosphatase levels, enzymes that have been described as regeneration markers.

Multiple forms of DNA polymerase from the thermo-acidophilic eubacterium Bacillus acidocaldarius: purification, biochemical characterization and possible biological role.

Two DNA polymerase isoenzymes, called DpA and DpB on the basis of their elution order from DEAE cellulose, were purified to homogeneity from the thermo-acidophilic eubacterium Bacillus acidocaldarius. The enzymes are weakly acidophilic proteins constituted by a single subunit of 117 and 103 kDa respectively. DpA and DpB differ in thermostability, in thermophilicity, in sensitivity to assay conditions and in resistance to sulphydryl-group blocking agents such as N-ethylmaleimide and p-hydroxymercuriobenzoate. They differ also in synthetic template-primer utilization, in the apparent Km for dNTPs and in processivity. In particular, DpA utilizes more effic iently synthetic templates-primers such as poly(dA).poly(dT), poly(dT). (rA)12-18 and poly(rA).(dT)12-18 and presents a greater tendency to accept dNTP analogues modified in the sugar or in the base ring, such as cytosine beta-d-arabinofuranoside 5'-triphosphate, 2',3'-dideoxyribonucleosides 5'-triphosphate, butylphenyl-dGTP and digoxigenin-conjugated dUTP. In addition, DpA presents an exonuclease activity that preferentially hydrolyses DNA in the 5'-3' direction, whereas DpB lacks this activity. The possible biological role of the enzymes is discussed.

Inhibition mechanisms of HIV-1, Mo-MuLV and AMV reverse transcriptases by Kelletinin A from Buccinulum corneum.

Kelletinin A [ribity pentakis (p-hydroxybenzoate)] (KA), an inhibitor of HTLV-1 replication isolated from Buccinulum corneum, showed a noncompetitive inhibitory activity with respect to the template primer and to dTTP in the poly(rA).oligo(dT)12-18-directed reaction of HIV-1, Mo-MuLV and AMV reverse transcriptases (RT). Analysis of natural and synthetic KA-related compounds showed that the inhibitory activity was strictly related to the structural peculiarities of the molecule. In the presence of DNA as template primer the inhibition mechanism was drastically modified: HIV-1 RT activity was stimulated by low concentrations of KA and was inhibited by increasing the concentration of the compound, while Mo-MuLV and AMV activities were irreversibly inhibited by the formation of a non-reactive complex. The RNase H activities of these RTs were not affected by KA. The results of this study suggest a different mechanism of interaction of Kelletinins with HIV-1 RT compared with other non-nucleoside inhibitors. A possible use of these drugs in combination therapy and in the design of structure-based reverse transcriptase inhibitors is discussed.

Antimitotic and antiviral activities of Kelletinin A in HTLV-1 infected MT2 cells.

Kelletinin A [ribityl-pentakis (p-hydroxybenzoate)] (KA), a natural compound isolated from the marine gastropod Buccinulum corneum, showed antiviral activity on the human T-cell leukemia virus type-1 (HTLV-1) and antimitotic activity on HTLV-1-infected MT2 cells. KA inhibited cellular DNA and RNA synthesis, without influencing protein synthesis, and interfered with viral transcription by reducing the levels of high molecular weight transcripts. Finally, the compound inhibited HTLV-1 reverse transcriptase in vitro.

A human factor that recognizes DNA substituted with 2-chloroadenine, an antileukemic purine analog.

2-Chloro-2'-deoxyadenosine (cladribine), an analog of deoxyadenosine, is an important new drug for the treatment of hairy cell leukemia and other forms of adult and pediatric leukemia. By a gel-shift binding assay, we identified an activity in HeLa nuclear extracts that recognizes and binds to oligonucleotides substituted with 2-chloroadenine (ClAde). The activity was specific for ClAde residues because control oligomers did not readily compete out the complex. The binding factor was a monomeric protein that was resistant to inactivation by heating at 45 degrees C but sensitive to heating at 65 degrees C, proteinase K treatment, and 5 mM ZnCl2. This protein, designated ClAde recognition protein (CARP), appeared to be related to a protein that recognized other forms of DNA damage. Gel-shift binding reactions with ultraviolet (UV)-irradiated oligomers revealed a UV-specific protein/DNA complex that had an electrophoretic mobility similar to that of the CARP/DNA complex, and CARP binding to ClAde-containing oligomers was readily competed out by UV-irradiated DNA. Moreover, CARP activity was present in extracts prepared from UV-sensitive xeroderma pigmentosum group A cells but not in a subset of cells from group E, suggesting that CARP was similar to a previously described repair associated factor, xeroderma pigmentosum-E binding factor. Our findings support a possible repair process for ClAde residues incorporated into cellular DNA.

2-Chloro-2'-deoxyadenosine monophosphate residues in DNA enhance susceptibility to 3'-->5' exonucleases.

2-Chloro-2'-deoxyadenosine triphosphate, a purine nucleotide analogue and potent antileukaemic agent, was incorporated into double-stranded 36-mers in place of dATP to investigate the effects of 2-chloroadenine (ClAde) on DNA polymerase-associated 3'-->5' exonuclease activity. ClAde residues within one strand of duplex DNA did not inhibit exonuclease activity; on the contrary, ClAde-containing minus strands were digested to a greater extent than was control DNA in the absence of deoxyribonucleoside triphosphates by Escherichia coli Klenow fragment, yeast DNA polymerase II and T4 DNA polymerase. After a 30 min incubation with 5 units of Klenow fragment, approximately 65% of control DNA remained in DNA fragments of 26 bases or larger compared with only approximately 25% of ClAde-substituted substrates. Unsubstituted plus strands opposite a ClAde-containing strand were likewise digested more quickly by 3'-->5' exonuclease, but only in the vicinity of the ClAde sites. Approx. 63% of the plus strands from ClAde-containing oligomers were less than 24 bases in length after a 25 min digestion period with Klenow fragment compared with only approximately 32% of control DNA. Such results indicate that, unlike other base modifications such as pyrimidine dimers, methoxy psoralen adducts and certain nucleoside analogues, all of which inhibit or decrease the rate of strand degradation by 3'-->5' exonucleases, incorporated ClAde enhances strand degradation of duplex DNA.

Template 2-chloro-2'-deoxyadenosine monophosphate inhibits in vitro DNA synthesis.

2'-Chloro-2'-deoxyadenosine triphosphate (cladribine), a purine nucleotide analog and potent antileukemic agent, was enzymatically incorporated into 98-base oligomers in place of dATP to investigate the molecular consequences of 2-chloroadenine (CIAde) in DNA. We have used the resultant oligomers as templates for purified DNA polymerases, to compare the rate and extent of in vitro DNA synthesis; the sites of polymerase pausing, if any; and the effects of increasing deoxyribonucleoside triphosphate (dNTP) concentrations on synthetic reactions. Compared with control template, CIAde-containing DNA strikingly reduced the overall amount and rate of chain elongation by human polymerase beta and Klenow fragment. Distinct pause sites, which were polymerase dependent, occurred primarily one or two bases before or just after nucleotide incorporation opposite template CIAde. Human polymerase alpha and phage T4 DNA polymerase likewise exhibited reduced synthesis on CIAde-substituted templates. Bypassing of CIAde residues was possible only at higher dNTP concentrations, with approximately 20- and 50-fold greater dNTP concentrations being required for synthesis beyond CIAde sites, compared with adenine residues, by polymerase alpha and beta, respectively. These results suggest that CiAde residues located within cellular template DNA may inhibit daughter strand synthesis and thus contribute to the cytotoxic effects of the drug.

Polymerase chain reaction amplification of single-stranded DNA containing a base analog, 2-chloradenine.

By utilization of polymerase chain reaction techniques, single-stranded DNA of defined length and sequence containing a purine analog, 2-chloroadenine, in place of adenine was synthesized. This was accomplished by a combination of standard polymerase chain amplification reactions with Thermus aquaticus DNA polymerase in the presence of four normal deoxynucleoside triphosphates, M13 duplex DNA as template, and two primers to generate double-stranded DNA 118 bases in length. An asymmetric polymerase chain reaction, which produced an excess of single-stranded 98-base DNA, was then conducted with 2-chloro-2'-deoxy-adenosine 5'-triphosphate in place of dATP and with only one primer that annealed internal to the original two primers. Standard polymerase chain reaction techniques alone conducted in the presence of the analog as the fourth nucleotide did not produce duplex DNA that was modified within both strands. This asymmetric technique allows the incorporation of an altered nucleotide at specific sites into large quantities of single-stranded DNA without using chemical phosphoramidite synthesis procedures and circumvents the apparent inability of DNA polymerase to synthesize fully substituted double-stranded DNA during standard amplification reactions. The described method will permit the study of the effects of modified bases in template DNA on a variety of protein-DNA interactions and enzymes.

Kelletinin I and kelletinin A from the marine mollusc Buccinulum corneum are inhibitors of eukaryotic DNA polymerase alpha.

The inhibitory effect of esters of p-hydroxybenzoic acid (kelletinins I and A), extracted from the marine gastropod Buccinulum corneum, have been tested on eukaryotic and prokaryotic enzymes of DNA metabolism such as DNA polymerases alpha and beta, DNA polymerase I, Exo III, pancreatic DNAse I, micrococcal DNAse and E. coli RNA polymerase. Kelletinin I and kelletinin A inhibit preferentially DNA polymerase alpha. The inhibitory effect of kelletinin I involves the hydroxyl group of p-hydroxybenzoic acid.

Replicating premeiotic germ cells of the mouse contain a novel DNA primase stimulatory factor.

A protein factor that stimulates DNA primase activity associated with DNA polymerase alpha has been identified in mouse germ cell populations enriched in spermatogonia and preleptotene spermatocytes. The partially purified factor enhances DNA primase activity from homologous cell types as well as DNA primase activity from Xenopus laevis oocytes in a poly dT or M 13 directed reaction. The factor does not stimulate DNA polymerase alpha activity in a gapped salmon sperm or poly dT-rA directed reaction. The DNA primase stimulating factor is identified in a male mouse germ cell population enriched in premeiotic cells; it is not detectable in middle-late pachytene spermatocytes, spermatids, Sertoli cells or fibroblasts.

DNA repair synthesis in mouse spermatogenesis involves DNA polymerase beta activity.

The role of DNA polymerase alpha-DNA primase complex and DNA polymerase beta in DNA replication and ultraviolet-induced DNA repair synthesis has been analyzed in mouse spermatogenesis. Autoradiographic experiments with germ cells in culture, indicating an involvement of DNA polymerase alpha and/or delta in DNA replication, and of DNA polymerase beta in DNA repair synthesis, have been confirmed by studying partially purified enzymes. These findings support the idea that, different from other biological systems, in meiotic and post meiotic male mouse germ cells DNA polymerase beta is the main DNA polymerase form needed for DNA repair.

DNA repair synthesis-related enzymes during spermatogenesis in the mouse.

To assess whether uracil DNA glycosylase and dUTP nucleotidohydrolase (dUTPase) can be involved in repair-type DNA synthesis associated to crossing-over or induced by UV and X-ray treatments, we have studied these enzyme activities in male mouse germ cells at specific stages of differentiation. Although the highest uracil DNA glycosylase activity was observed in dividing germ cells (spermatogonia and preleptotene spermatocytes), some activity was also detected in meiotic (3.5%) and post-meiotic (1.0%) cells with a relative maximum of activity at pachytene stage (4.7%) when meiotic crossing-over takes place. These findings suggest that uracil DNA glycosylase is involved, in this biological system, in DNA replication and in repair-type DNA synthesis. dUTPase is present at all the stages of spermatogenesis studied but, unlike thymidylate synthetase which is mainly associated with replicating germ cells, dUTPase activity is maximal in spermatocytes at pachytene stages. The data reported suggest that, in this biological system, the main role of dUTPase is to degrade dUTP to prevent misincorporation of uracil into DNA during crossing-over, rather than to participate in the biosynthetic pathway of dTTP.