The midgut epithelium of mosquitoes adjusts cell proliferation and endoreplication to respond to physiological challenges.

BACKGROUND: Hematophagous mosquitoes transmit many pathogens that cause human diseases. Pathogen acquisition and transmission occur when female mosquitoes blood feed to acquire nutrients for reproduction. The midgut epithelium of mosquitoes serves as the point of entry for transmissible viruses and parasites. RESULTS: We studied midgut epithelial dynamics in five major mosquito vector species by quantifying PH3-positive cells (indicative of mitotic proliferation), the incorporation of nucleotide analogs (indicative of DNA synthesis accompanying proliferation and/or endoreplication), and the ploidy (by flow cytometry) of cell populations in the posterior midgut epithelium of adult females. Our results show that the epithelial dynamics of post-emergence maturation and of mature sugar-fed guts were similar in members of the Aedes, Culex, and Anopheles genera. In the first three days post-emergence, ~ 20% of cells in the posterior midgut region of interest incorporated nucleotide analogs, concurrent with both proliferative activity and a broad shift toward higher ploidy. In mature mosquitoes maintained on sugar, an average of 3.5% of cells in the posterior midgut region of interest incorporated nucleotide analogs from five to eight days post-emergence, with a consistent presence of mitotic cells indicating constant cell turnover. Oral bacterial infection triggered a sharp increase in mitosis and nucleotide analog incorporation, suggesting that the mosquito midgut undergoes accelerated cellular turnover in response to damage. Finally, blood feeding resulted in an increase in cell proliferation, but the nature and intensity of the response varied by mosquito species and by blood source (human, bovine, avian or artificial). In An. gambiae, enterocytes appeared to reenter the cell cycle to increase ploidy after consuming blood from all sources except avian. CONCLUSIONS: We saw that epithelial proliferation, differentiation, and endoreplication reshape the blood-fed gut to increase ploidy, possibly to facilitate increased metabolic activity. Our results highlight the plasticity of the midgut epithelium in mosquitoes' physiological responses to distinct challenges.

N-(phosphonacetyl)-L-aspartate induces TAp73-dependent apoptosis by modulating multiple Bcl-2 proteins: potential for cancer therapy.

p53 is essential for the cellular responses to DNA damage that help to maintain genomic stability. However, the great majority of human cancers undergo disruption of the p53-network. Identification and characterization of molecular components important in both p53-dependent and -independent apoptosis might be useful in developing novel therapies for cancers. In the complete absence of p53, cells treated with N-(phosphonacetyl)-L-aspartate (PALA) continue to synthesize DNA slowly and eventually progress through S-phase, suffering severe DNA damage that in turn triggers apoptosis, whereas cells with functional p53 undergo growth arrest. In this study, we investigated apoptotic signaling in response to PALA and the role of p53 expression in this pathway. We found that treatment of cells lacking p53 with PALA induced TAp73, Noxa and Bim and inactivation of these proteins with dominant-negative plasmids or small interfering RNAs significantly inhibited apoptosis, suggesting that PALA-induced apoptosis was mediated via TAp73-dependent expression of Noxa and Bim. However, PALA treatment inhibited the expression of ΔNp73 only in cells lacking p53 but not in cells expressing p53. In addition, PALA treatment inhibited Bcl-2, and overexpression of Bcl-2 significantly inhibited PALA-induced apoptosis. Moreover, expression of p53 in these cells protected them from PALA-induced apoptosis by activating p21, sustaining the expression of ΔNp73 and inhibiting the induction of Noxa and Bim. Taken together, our study identifies novel but opposing roles for the p53 and TAp73 in the induction of Noxa and Bim and regulation of apoptosis. Our data will help to develop strategies to eliminate cancer cells lacking p53 while protecting normal cells with wild-type p53.

Regulation of p53 expression by the RAS-MAP kinase pathway.

Activation of MAP kinase leads to the activation of p53-dependent pathways, and vice-versa. Although the amount of p53 protein increases in response to MAP kinase-dependent signaling, the basis of this increase is not yet fully understood. We have isolated the mutant cell line AP14, defective in p53 expression, from human HT1080 fibrosarcoma cells, which have an activated ras allele. The expression of p53 mRNA and protein is approximately 10-fold lower in AP14 cells than in the parental cells. The high constitutive phosphorylation and activities of the MAP kinases ERK1 and ERK2 in HT1080 cells are greatly reduced in AP14 cells, although the levels of these proteins are unchanged, suggesting that the defect in the mutant cells affects the steady-state phosphorylation of ERK1 and ERK2. Overexpression of ERK2 in AP14 cells restored both MAP kinase activity and p53 expression, and incubation of the mutant cells with the phosphatase inhibitor orthovanadate resulted in strong coordinate elevation of MAP kinase activity and p53 expression. The levels of expression of the p53-regulated gene p21 parallel those of p53 throughout, showing that basal p21 expression depends on p53. The levels of p53 mRNA increased by 5-8-fold when activated ras was introduced into wild-type cells, and the levels of the p53 and p21 proteins decreased substantially in wild-type cells treated with the MEK inhibitor U0216. We conclude that MAP kinase-dependent pathways help to regulate p53 levels by regulating the expression of p53 mRNA.

Mechanisms of G2 arrest in response to overexpression of p53.

Overexpression of p53 causes G2 arrest, attributable in part to the loss of CDC2 activity. Transcription of cdc2 and cyclin B1, determined using reporter constructs driven by the two promoters, was suppressed in response to the induction of p53. Suppression requires the regions -287 to -123 of the cyclin B1 promoter and -104 to -74 of the cdc2 promoter. p53 did not affect the inhibitory phosphorylations of CDC2 at threonine 14 or tyrosine 15 or the activity of the cyclin-dependent kinase that activates CDC2 by phosphorylating it at threonine 161. Overexpression of p53 may also interfere with the accumulation of CDC2/cyclin B1 in the nucleus, required for cells to enter mitosis. Constitutive expression of cyclin B1, alone or in combination with the constitutively active CDC2 protein T14A Y15F, did not reverse p53-dependent G2 arrest. However, targeting cyclin B1 to the nucleus in cells also expressing CDC2 T14A Y15F did overcome this arrest. It is likely that several distinct pathways contribute to p53-dependent G2 arrest.

p53 inhibits entry into mitosis when DNA synthesis is blocked.

Human and mouse fibroblasts with normal p53 fail to enter mitosis when DNA synthesis is blocked by aphidicolin or hydroxyurea. Isogenic p53-null fibroblasts do enter mitosis with incompletely replicated DNA, revealing that p53 contributes to a checkpoint that ensures that mitosis does not occur until DNA synthesis is complete. When treated with N-(phosphonacetyl)-L-aspartate (PALA), which inhibits pyrimidine nucleotide synthesis, leading to synthesis of damaged DNA from highly unbalanced dNTP pools, p53-null cells enter mitosis after they have completed DNA replication, but cells with wild-type p53 do not, revealing that p53 also mediates a checkpoint that monitors the quality of newly replicated DNA.

A p53-dependent S-phase checkpoint helps to protect cells from DNA damage in response to starvation for pyrimidine nucleotides.

Normal mammalian cells arrest primarily in G1 in response to N-(phosphonacetyl)-L-aspartate (PALA), which starves them for pyrimidine nucleotides, and do not generate or tolerate amplification of the CAD gene, which confers resistance to PALA. Loss of p53, accompanied by loss of G1 arrest, permits CAD gene amplification and the consequent formation of PALA-resistant colonies. We have found rat and human cell lines that retain wild-type p53 but have lost the ability to arrest in G1 in response to PALA. However, these cells still fail to give PALA-resistant colonies and are protected from DNA damage through the operation of a second checkpoint that arrests them reversibly within S-phase. This S-phase arrest, unmasked in the absence of the G1 checkpoint, is dependent on p53 and independent of p21/waf1.

MYC abrogates p53-mediated cell cycle arrest in N-(phosphonacetyl)-L-aspartate-treated cells, permitting CAD gene amplification.

Genomic instability, including the ability to undergo gene amplification, is a hallmark of neoplastic cells. Similar to normal cells, "nonpermissive" REF52 cells do not develop resistance to N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of the synthesis of pyrimidine nucleotides, through amplification of cad, the target gene, but instead undergo protective, long-term, p53-dependent cell cycle arrest. Expression of exogenous MYC prevents this arrest and allows REF52 cells to proceed to mitosis when pyrimidine nucleotides are limiting. This results in DNA breaks, leading to cell death and, rarely, to cad gene amplification and PALA resistance. Pretreatment of REF52 cells with a low concentration of PALA, which slows DNA replication but does not trigger cell cycle arrest, followed by exposure to a high, selective concentration of PALA, promotes the formation of PALA-resistant cells in which the physically linked cad and endogenous N-myc genes are coamplified. The activated expression of endogenous N-myc in these pretreated PALA-resistant cells allows them to bypass the p53-mediated arrest that is characteristic of untreated REF52 cells. Our data demonstrate that two distinct events are required to form PALA-resistant REF52 cells: amplification of cad, whose product overcomes the action of the drug, and increased expression of N-myc, whose product overcomes the PALA-induced cell cycle block. These paired events occur at a detectable frequency only when the genes are physically linked, as cad and N-myc are. In untreated REF52 cells overexpressing N-MYC, the level of p53 is significantly elevated but there is no induction of p21waf1 expression or growth arrest. However, after DNA is damaged, the activated p53 executes rapid apoptosis in these REF52/N-myc cells instead of the long-term protective arrest seen in REF52 cells. The predominantly cytoplasmic localization of stabilized p53 in REF52/N-myc cells suggests that cytoplasmic retention may help to inactivate the growth-suppressing function of p53.

Defective induction but normal activation and function of p53 in mouse cells lacking poly-ADP-ribose polymerase.

Poly-ADP-ribose polymerase (PARP) and p53 are both induced by DNA damage and each has been proposed to mediate the normal cellular response to damage. We find that embryo fibroblasts from PARP-null mice have a approximately twofold lower basal level of p53 and that the induction of p53 in response to DNA damage or nucleotide depletion is more than twofold less than in normal mouse cells. These factors combine to decrease the induced level of the p53 protein in PARP-deficient cells by 4-5-fold, compared to normal cells. However, there is virtually no decrease in the induction of p53 activity in PARP-deficient cells, as assayed with a p53-responsive promoter. Furthermore, cells lacking PARP arrest normally in G1 after DNA damage, in contrast to cells lacking p53, where this checkpoint is absent. Other p53-dependent properties, such as the mitotic spindle checkpoint and permissivity for gene amplification, are also normal in PARP-deficient cells. We conclude that the induced level of the p53 protein is governed by a combination of PARP-dependent and PARP-independent pathways and that the activation of p53 is largely PARP-independent. The results are consistent with a model in which the regulation of gene expression by p53 involves both increases in the amount of the protein and activation of p53 as a transcription factor.

Multiple mechanisms of N-phosphonacetyl-L-aspartate resistance in human cell lines: carbamyl-P synthetase/aspartate transcarbamylase/dihydro-orotase gene amplification is frequent only when chromosome 2 is rearranged.

Rodent cells resistant to N-phosphonacetyl-L-aspartate (PALA) invariably contain amplified carbamyl-P synthetase/aspartate transcarbamylase/dihydro-orotase (CAD) genes, usually in widely spaced tandem arrays present as extensions of the same chromosome arm that carries a single copy of CAD in normal cells. In contrast, amplification of CAD is very infrequent in several human tumor cell lines. Cell lines with minimal chromosomal rearrangement and with unrearranged copies of chromosome 2 rarely develop intrachromosomal amplifications of CAD. These cells frequently become resistant to PALA through a mechanism that increases the aspartate transcarbamylase activity with no increase in CAD copy number, or they obtain one extra copy of CAD by forming an isochromosome 2p or by retaining an extra copy of chromosome 2. In cells with multiple chromosomal aberrations and rearranged copies of chromosome 2, amplification of CAD as tandem arrays from rearranged chromosomes is the most frequent mechanism of PALA resistance. All of these different mechanisms of PALA resistance are blocked in normal human fibroblasts.

Induction of fragility at the human RNU2 locus by cytosine arabinoside is dependent upon a transcriptionally competent U2 small nuclear RNA gene and the expression of p53.

Chromosomal fragile sites are regions that are intrinsically unstable and are susceptible to experimentally induced damage. In most cases, the target and mechanism of induction of fragility are unknown. Using ectopic integration of engineered DNA arrays to create "new" fragile sites, we and others have previously shown that the transcriptionally competent U2 gene is necessary and sufficient for induction of fragility at the RNU2 locus upon infection of human cells with Adenovirus 12. In the present study we have investigated the response of the RNU2 locus to cytosine arabinoside (araC), an inhibitor of DNA polymerases and a common inducer of fragile sites. We demonstrate that the RNU2 locus is sensitive to the drug and that araC-induced fragility is dependent upon a functional U2 gene and on the expression of the cellular p53 protein. Our results identify a novel DNA structure associated with fragile sites and suggest a role for transcription and repair processes in RNU2 fragility.

The induction of partial resistance to photodynamic therapy by the protooncogene BCL-2.

Photodynamic therapy (PDT) is an efficient inducer of apoptosis, an active form of cell death that can be inhibited by the BCL-2 oncoprotein. The ability of BCL-2 to modulate PDT-induced apoptosis and overall cell killing has been studied in a pair of Chinese hamster ovary cell lines that differ from one another by a transfected human BCL-2 gene in one of them (Bissonnette et al, Nature 359, 552-554, 1992). Cells were exposed to the phthalocyanine photosensitizer Pc 4 and various fluences of red light. Pc 4 uptake was identical in the two cell lines. The parental cells displayed a high incidence of apoptosis after PDT, whereas at each fluence there was a much lower incidence of apoptosis in the BCL-2-expressing cells. Apoptosis was monitored by (a) observation of 50 kbp and oligonucleosome-size DNA fragments by gel electrophoresis, (b) flow cytometry of cells labeled with fluorescently tagged dUTP by terminal deoxynucleotidyl transferase and (c) fluorescence microscopy of acridine orange-stained cells. The time course of apoptosis varied with the PDT dose, suggesting that only after moderately high doses (> 99% loss of clonogenicity) was there a relatively synchronous and rapid entry of many cells into apoptosis. At PDT doses reducing cell survival by 90 or 99%, significant increases in apoptotic cells were found in the population after 6-12 h. Clonogenic assays showed that BCL-2 protein inhibited not only apoptosis but overall cell killing as well, effecting a two-fold resistance at the 10% survival level. Thus, BCL-2-expressing cells may be relatively resistant to PDT.

The role of p53 in regulating genomic stability when DNA and RNA synthesis are inhibited.

In addition to its induction by DNA damage, p53 is induced by drugs that starve cells for DNA and RNA precursors, or by inhibitors of DNA or RNA polymerase. In normal cells, the induction of p53 by dNTP starvation serves a protective role, mediating rapid, reversible cell-cycle arrest without DNA damage. In most cell lines, this first line of defense is missing, so that starvation for dNTPs causes DNA to break, thus increasing the probability of genomic instability, chromosome deletions and gene amplification. The mechanism of how p53 is induced remains unclear.

p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts.

Increased expression of wild-type p53 in response to DNA damage arrests cells late in the G1 stage of the cell cycle by stimulating the synthesis of inhibitors of cyclin-dependent kinases, such as p21/WAF1. To study the effects of p53 without the complication of DNA damage, we used tetracycline to regulate its expression in MDAH041 human fibroblasts that lack endogenous p53. When p53 is expressed at a level comparable to that induced by DNA damage in other cells, most MDAH041 cells arrested in G1, but a significant fraction also arrested in G2/M. Cells released from a mimosine block early in S phase stopped predominantly in G2/M in the presence of p53, confirming that p53 can mediate arrest at this stage, as well as in G1. In these cells, there was appreciable induction of p21/WAF1. MDAH041 cells arrested by tetracycline-regulated p53 for as long as 20 days resumed growth when the p53 level was lowered, in striking contrast to the irreversible arrest mediated by DNA damage. Therefore, irreversible arrest must involve processes other than or in addition to the interaction of p53-induced p21/WAF1 with G1 and G2 cyclin-dependent kinases.

Elevation of GRP-78 and loss of HSP-70 following photodynamic treatment of V79 cells: sensitization by nigericin.

Chinese hamster V79 cells were treated with photodynamic therapy (PDT) sensitized by aluminum phthalocyanine (AlPc) or with the ionophore nigericin or with combinations of PDT and nigericin. We previously showed that PDT and nigericin interact synergistically in the killing of these cells; i.e. doses of PDT that kill no more than 10% of the cells in combination with nontoxic exposures to nigericin lead to a loss of clonogenicity of three to five orders of magnitude. Photodynamic therapy induces an enhanced rate of expression of the stress gene grp-78 both at the transcriptional and translational levels and causes a decrease in the synthesis of the constitutive heat shock protein HSP-70 as well as in expression of HSP-70 mRNA. When the cells are exposed to PDT in the presence of nigericin, these effects are elicited at three- to four-fold lower PDT doses. Thus, PDT in the presence of nigericin is much more effective in inducing the changes in gene expression than is PDT alone. In the absence of nigericin the PDT dose inducing a two-fold increase in GRP-78 accumulation causes little or no loss of clonogenicity. In the presence of nigericin, however, the PDT dose leading to a similar change in GRP-78 level produces up to a 50% loss of clonogenicity. The fact that nigericin is dose-modifying for both cell killing and stress responses suggests that nigericin either increases the yield of oxidative damage from a given dose of PDT or magnifies the cellular response to a constant level of oxidative stress.

Regulation and mechanisms of gene amplification.

Amplification in rodent cells usually involves bridge-breakage-fusion (BBF) cycles initiated either by end-to-end fusion of sister chromatids, or by chromosome breakage. In contrast, in human cells, resistance to the antimetabolite N-(phosphonacetyl)-L-aspartate (PALA) can be mediated by several different mechanisms that lead to overexpression of the target enzyme carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase (CAD). Mechanisms involving BBF cycles account for only a minority of CAD amplification events in the human fibrosarcoma cell line HT 1080. Here, formation of a 2p isochromosome and overexpression of CAD by other types of amplification events (and even without amplification) are much more prevalent. Broken DNA is recognized by mammalian cells with intact damage-recognition pathways, as a signal to arrest or to die. Loss of these pathways by, for example, loss of p53 or pRb tumour suppressor function, or by increased expression of ras and myc oncogenes, causes non-permissive rat and human cells to become permissive both for amplification and for other manifestations of DNA damage. In cells that are already permissive, amplification can be stimulated by overexpressing oncogenes such as c-myc or ras, or by damaging DNA in a variety of ways. To supplement genetic analysis of amplification in mammalian cells, an amplification selection has been established in Schizosaccharomyces pombe. Selection with LiCl yields cells with amplified sod2 genes in structures related to those observed in mammalian cells. The effect on amplification in S. pombe can now be tested for any mutation in a gene involved in repair of damaged DNA or in normal cellular responses to DNA damage.

Phospholipase activation triggers apoptosis in photosensitized mouse lymphoma cells.

Photodynamic therapy (PDT), an experimental cancer treatment employing a photosensitizer and visible light, is a highly efficient inducer of apoptosis (or programmed cell death) in mouse L5178Y lymphoma cells, resulting in extensive DNA fragmentation within 1-2 h. The major targets for PDT are in cellular membranes, and we now find that PDT sensitized by aluminum phthalocyanine causes the rapid (< 1 min) activation of phospholipase C and the breakdown of membrane phosphoinositides, as well as a similarly rapid release of Ca2+ from intracellular pools. A phospholipase C inhibitor, U73122, blocks the rapid transient increases in both inositol-1,4,5-trisphosphate and intracellular Ca2+ levels as well as the subsequent fragmentation of nuclear DNA, whereas the analogue U73343 is much less effective against all of the aforementioned responses. In addition, p-bromphenacyl bromide, an inhibitor of phospholipase A2, blocks DNA fragmentation, and PDT stimulates the release of arachidonic acid, probably by phospholipase A2-dependent breakdown of membrane phospholipids. Thus, photodynamic damage to cell membranes can mimic natural stimuli of phospholipases and initiate apoptosis in L5178Y cells.

The flax ribosomal RNA-encoding genes are arranged in tandem at a single locus interspersed by 'non-rDNA' sequences.

The ribosomal RNA (rRNA)-encoding genes (rDNA) in flax, estimated to be present in about 2400 copies per diploid nucleus, have been reported as a single homogeneous repeat unit of 8.6 kb. In situ hybridization analysis indicated that these genes were located at a single site on one pair of chromosomes. However, an analysis of a flax variety, CI 1303, has revealed heterogeneity in the intergenic spacer of the rDNA repeat unit. A genetic analysis of rDNA inheritance in two flax lines, Stormont Cirrus and CI 1303, has again supported the observation that there is a single rDNA locus in this plant species. Screening of four different genomic libraries made in methylation-sensitive and -insensitive systems, and the analysis of 40 phage clones, demonstrate a much higher number than that expected of junctions between rDNA and non-rDNA. Direct evidence of rRNA-encoding genes being present in tandem comes from a few phage clones that contain more than two rDNA repeats. The evidence presented here indicates that rDNA, although present at a single locus in tandem arrays, may be interrupted frequently by other non-rDNA sequences, thus giving rise to questions about their organization into long tandem arrays.

Photodynamic therapy induces rapid cell death by apoptosis in L5178Y mouse lymphoma cells.

The mode of cell death of two strains of mouse lymphoma L5178Y cells was studied following photodynamic therapy (PDT) sensitized by chloroaluminum phthalocyanine. Strains LY-R and LY-S differ in their relative sensitivities to UVC radiation, X-radiation, and PDT; both responded to PDT by undergoing apoptosis. The DNA was degraded into fragments with lengths which are multiples of approximately 180-190 base pairs (i.e., oligonucleosome size), a biochemical marker of apoptosis. The DNA fragmentation was dose and time dependent which indicates this response to be an enzymic process related to cell killing. Cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor, enhanced the endonucleolytic DNA fragmentation. Transmission electron microscopy revealed chromatin condensation around the periphery of the nucleus, which is also characteristic of apoptosis. The induction of apoptosis in L5178Y cells by PDT was rapid, with marked degradation of DNA occurring in as little as 30 min. The rapidity of the response to PDT suggests that cellular damage produced by PDT can directly activate endonucleolysis and chromatin condensation, thereby by-passing many of the early steps in the signal transduction program which are acted upon by other agents causing apoptosis.

The ubiquitin-encoding multigene family of flax, Linum usitatissimum.

Ubiquitin (Ubq), a 76-amino acid (aa) protein, is found in all eukaryotic organisms and is one of the most conserved proteins so far studied. It is implicated in many cellular processes. The Ubq-encoding genes (ubq) are generally present as a multigene family. In flax, we have estimated that this multigene family contains at the most ten members. The initial flax ubq sequences were isolated from a flax genomic library in lambda EMBL4 using a heterologous Arabidopsis thaliana ubq probe. An 916-bp fragment from one of the phage clones was subcloned and sequenced. The aa sequence derived from the nucleotide sequence of this fragment is identical to that of other plant Ubqs. This fragment was then used to isolate additional flax ubq clones. In all, eleven phage lambda clones, which represent six members of the gene family, were restriction-mapped and characterized. These six members are represented as three monomers, three poly-Ubqs, one hexamer and two tetramers. They can be present at either a single locus (two of the monomers and one of the poly-Ubqs) or at two loci (the remaining three genes). The other four members of the family are yet to be cloned and characterized.