Snail misexpression leads to hyperproliferation and a reduction in intercellular adhesion. When E-cadherin is transcriptionally down-regulated, associated adhesion proteins with dual functions in signaling are released from cell-cell contacts, a process which we demonstrate leads to Ras-MAPK activation. These studies provide insights into how multipotent cells within a sheet are stimulated to undergo transcriptional changes that result in proliferation, junctional remodeling, and bud formation. This novel signaling pathway further weaves together the web of different morphogens and downstream transcriptional events that guide hair bud formation within the developing skin.In a common theme of organogenesis, certain cells within a multipotent epithelial sheet exchange signals with their neighbors and develop into a bud structure. Using hair bud morphogenesis as a paradigm, we employed mutant mouse models and cultured keratinocytes to dissect the contributions of multiple extracellular cues in orchestrating adhesion dynamics and proliferation to shape the cluster of cells involved. We found that transforming growth factor β2 signaling is necessary to transiently induce the transcription factor Snail and activate the Ras-mitogen-activated protein kinase (MAPK) pathway in the bud. In the epidermis,  The study of hair follicle morphogenesis provides insights into how cells within a sheet can be triggered to proliferate, remodel, and form buds - a recurring theme in development Mammalian development involves the morphogenesis of complex three-dimensional structures from seemingly uniform sheets or masses of cells. A simple bud-like structure initiates the formation of many organs, including lungs, spinal cord, mammary glands, and hair follicles . The mulAt the helm of these molecular communication pathways are Wnts, bone morphogenic proteins (BMPs), transforming growth factor βs (TGF-βs), and fibroblast growth factors (FGFs). Through activation of cell surface transmembrane receptors, these external signaling molecules trigger distinct cascades of intracellular events that culminate in changes in gene expression, growth, and differentiation . How thiThe development of a bud requires a number of coordinated changes in the behavior of the targeted cells within an epithelial sheet. The process must be accompanied by alterations in the proliferation, polarity, shape, and adhesiveness of selected cells, as well as by modifications in their underlying basal lamina. Thus, extracellular epithelial-mesenchymal crosstalk must be intricately orchestrated to couple the determination of distinct cell fates with the contemporaneous remodeling of the physical and structural properties of the cell.Among the few dispensable organs, hair follicles offer an excellent model system to study epithelial bud formation. Mammalian skin epithelium begins as a single sheet of multipotent ectodermal cells. During development, specialized mesenchymal cells populate the skin in a spatially defined pattern to initiate the complex epithelial-mesenchymal crosstalk that will specify the bud . Once coPreviously, we delineated how two respective epithelial and mesenchymal signals, Wnts and the BMP-inhibitory factor noggin, function in concert to induce lymphoid enhancer factor-1/β-catenin (LEF-1/β-catenin)-mediated gene transcription within the follicle placode . The dowE-cadherin promoter activity, we were intrigued by the close proximity of the LEF-1/β-catenin binding site to a site known to bind the Snail/Slug family of zinc finger transcriptional repressor proteins [E-cadherin promoter activity [As we probed more deeply into the underlying mechanisms governing proteins ,13,14,15proteins ,17. Bud Snail gene expression in embryonic skin keratinocytes. In contrast, we provide in vitro, transgenic (Tg), and gene targeting evidence to show that TGF-β2 and small phenotype– and mothers against decapentaplegic–related protein 2 (SMAD2) signaling are upstream inducers of Snail gene expression in skin epithelium. In the absence of TGF-β2 signaling and Snail gene expression, hair placodes can form, but further follicle down-growth is blocked. Our studies point to the view that Snail likely functions downstream of cell fate specification, at a stage where the bud begins to exhibit enhanced proliferation and migration.In the present study, we discovered that Snail is expressed briefly at an early stage of hair bud formation, when E-cadherin down-regulation and activation of proliferation take place. Thereafter, Snail disappears and remains absent during subsequent follicle down-growth and maturation. This exquisite pattern appears to be functionally relevant since altering it in vivo correspondingly affects features associated with hair bud formation, including down-regulation of E-cadherin, increased proliferation, and repressed terminal differentiation. Although the temporal spike of Snail in the hair bud is reflected at the mRNA level and seems to follow Wnt signaling and BMP inhibition, LEF-1/β-catenin activation does not appear to induce E-cadherin expression in keratinocytes [Although Snail family members are most frequently associated with EMTs, they also participate in many malignant processes involving a down-regulation but not a quantitative abrogation of intercellular junctions . The raninocytes , coupledinocytes ,19, we tSnail mRNA expression during a period of skin embryogenesis when waves of hair follicles are forming (unpublished data).To pinpoint specifically where Snail mRNA is expressed in the developing skin, we conducted in situ hybridization using a cRNA probe unique to the Snail 3′ untranslated region (UTR). Embryonic day 17.5 (E17.5) was chosen, since the multiple waves of follicle morphogenesis occurring at this time enabled us to evaluate Snail expression at different stages of the process. As shown in Snail mRNA expression during follicle development was most apparent in hybridized skin sections containing follicles from two different waves of morphogenesis -tagged Snail protein . In this respect, the skin epithelium resembled that of the hair bud, where the down-regulation in junction proteins is permissive for cell-cell remodeling without abrogating intercellular adhesion.Snail Tg epidermis and hair buds extended to the hyperproliferative state, leading us to wonder whether the down-regulation of AJ proteins might contribute to this condition. Given the increase in pMAPK staining in Snail Tg epidermis (see The similarities between rmis see G, we usermis see D. CoexprSnail Tg epidermis was particularly intriguing since prior studies have reported cyclin D gene as a direct target of TCF/β-catenin transcription [Numerous studies have correlated a down-regulation of E-cadherin with a translocation of β-catenin to the nucleus and a transactivation of genes that are regulated by the LEF-1/T cell factor (TCF) family of DNA binding proteins ,24,25. Tcription . This sacription  gave no Snail Tg epidermis. In addition to its documented ability to bind α-catenin [Snail Tg mice but not from the corresponding wild-type (WT) animals  may be regulating the Snail gene. A variety of extracellular signals have an impact on the cell type-specific expression of different Snail family members, and many of them, including Wnts, BMPs, FGFs, and TGF-βs, also affect hair bud development [The temporal spike of elopment ,16,28. SE-cadherin promoter and induced a LEF-1/β-catenin-sensitive reporter gene, TOPFLASH [Snail expression in keratinocytes.Previously, we showed that effective transmission of a Wnt-3a signal in cultured keratinocytes can be achieved through their exposure to the BMP inhibitor noggin, which induces LEF-1 expression . In vitrTOPFLASH . In contTOPFLASH ,29,30, oTGF-β genes, only the TGF-β2 null state blocked follicle development at the hair bud stage [Snail expression in keratinocytes isolated from the basal layer of the epidermis. Though there is no cell culture system available to specifically study placodal cells, these keratinocytes are their progenitors and are the closest approximation available to study the behavior of epithelial cells of the placode.TGF-β1 has been shown to induce Snail family members in hepatocytes and heart , 31. In ud stage . Thus, wSnail expression could not be maintained (Snail expression correlated with phosphorylated SMAD2 (pSMAD2) induction, its decline seemed to rely on secondary downstream events.Interestingly, treatment of cultured keratinocytes with as little as 5 ng/ml of TGF-β2 caused a rapid and transient induction of Snail B. Followintained B. Thus, Snail promoter activity in keratinocytes might be sensitive to TGF-β2 signaling (Snail gene. At 2 d after transient transfection, keratinocytes were treated with TGF-β2 (t = 0) and then assayed for transgene activity over the same time course in which we had observed Snail protein induction. The results of this experiment are presented in The rapid kinetics of Snail expression were reflected at the mRNA level, suggesting that ignaling C. To tesSnail promoter activity had increased 3-fold, and by 2 h, it peaked to approximately 10-fold over control levels  family [Recent studies on hair bud morphogenesis suggest that Wnt signals likely from the epithelium and BMP inhibitory signals from the underlying mesenchyme converge to produce an active transcription factor complex involving β-catenin and LEF-1, which in turn plays a key role in specifying the hair follicle fate ,30,36,37e absent ,38. Thes) family ,40. FurtTGF-β1-null skin, which exhibits an extended growing phase of postnatal hair follicles, TGF-β2-null skin displays an embryonic block in follicle bud progression [TGF-β2 null buds appear unable to progress to the down-growth phase, a feature that cannot be explained readily on the basis of previously established effects of TGF-βs.TGF-βs are known to promote withdrawal of keratinocytes from the cell cycle . Hence, gression . Althouggression , about 5Our finding that TGF-β2 is upstream from Ki67 expression and MAPK activation lends further support to the notion that hair follicle keratinocytes at this early stage of development react to TGF-β2 signaling in a fashion opposite to that typically expected for TGF-β factors. This said, based upon pSMAD2 immunohistochemistry, the immediate steps of downstream signaling appeared to be intact. Thus, we surmise that the proliferative outcome is likely to be rooted in differences in the repertoire of activated SMAD target genes. In this regard, the positive effects of TGF-β2 on proliferation within the hair bud may be more analogous to what has been seen in progression of squamous cell carcinoma to metastatic carcinoma  rather tSnail gene as a potential target of TGF-β signaling [Snail gene expression that occurs in the developing hair bud. The additional correlation between epithelial hyperproliferation and Snail transgene expression further fostered our interest in a possible link between TGF-β2 and Snail. Our functional studies demonstrate that without TGF-β2, Snail expression is abolished in the mutant hair buds, and conversely, in K14-Smad2 skin, Snail is ectopically activated. Moreover, our in vitro studies indicate that even sustained TGF-β2 exposure may cause only a transient induction of Snail, offering a possible explanation as to why Snail is so briefly expressed during hair follicle morphogenesis. An additional point worth mentioning is that prolonged expression of Tg Snail in postnatal skin resulted in morphological and biochemical signs of epithelial to mesenchymal transitions (unpublished data), underscoring why transient Snail expression may be so important during normal hair follicle morphogenesis [The prior identification of the ignaling  was introgenesis .Snail Tg mice seemed indicative of a defect in follicle formation  cell lines, Snail has been shown to block cell cycle progression and promote motility and shape changes for invasion . While oIrrespective of these differences, our in vivo studies do not stand alone, as there are many situations in which a down-regulation in AJ proteins correlate with enhanced proliferation. In fact, a myriad of diverse mechanisms have been implicated in activating epithelial proliferation upon down-regulation of AJ proteins ,23,24,48Snail fused to GST (Covance, Princeton, New Jersey, United States). Recombinant human TGF-β2 was purchased from R&D (Minneapolis, Minnesota, United States). Heat inactivated TGF-β2 was generated by heating the recombinant protein at 100 °C for 10 min.Primary antibodies used were against: E-cadherin (M. Takeichi, Kyoto University, Japan); α-catenin, β-catenin, pMAPK, tubulin (Sigma, St. Louis, Missouri, United States), Ajuba (G. Longmore, Washington University, St. Louis, Missouri, United States); β4 integrin/CD104 , laminin 5 (R. Burgeson, Harvard University, Cambridge, Massachusetts, United States), K5, K1, loricrin (Fuchs Lab), involucrin, fillagrin , MAPK, pSMAD2 ; Grb-2 ; P-cadherin ; HA , vimentin , Ki67 (Novo Castra, Newcastle Upon Tyne, United Kingdom), keratin 6 , cyclin D , and TGF-β2 (L. Gold, New York University, New York, New York, United States). FITC-, Texas Red-, or HRP-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, Pennsylvania, United States). Biotinylated secondary antibodies were from Vector Labs . Dilutions were according to the manufacturer's recommendation. The Snail antibody was generated in Guinea pigs by inoculating them with the N-terminal sequence of murine K14-Snail Tg mouse was generated by digesting the pcDNA3-mm Snail-HA plasmid (G. de Herreros, Universitat Pompeu, Fabra, Barcelona, Spain) with BamHI and NotI and subcloned into the K14 vector [K14-Smad 2 Tg mouse was reported in Ito et al., 2001. The TGF-β2 knockout (KO) mouse was described in [shh KO mouse [The 4 vector . The linribed in . The shhKO mouse  and TOPGProtein extracts from primary keratinocytes were generated either by lysing cells in lysis buffer (1% NP-40, 1% sodium deoxycholate, 20 mM Tris-Cl [pH 7.4], 140 mM NaCl containing 1 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride, and protease inhibitors) or directly in Laemmli bβuffer and boiled. For skin tissue: Frozen tissue was pulverized in a liquid nitrogen-cooled Gevebesmascher and the powder scraped into a chilled microcentrifuge tube. RIPA buffer (1% Triton X-100 in PBS with 10 mM EDTA, 150 mN NaCl, 1% sodium deoxycholate, and 0.1% SDS) and protease inhibitors or Laemmli buffer was added. The cell suspension was sonicated three times for 15 s and centrifuged at 14,000 rpm at 4 °C. The supernatant was separated from the pellet and used in the experiments. Extracts subjected to immunoprecipitation were precleared with Protein G Sepharose (Amersham, Piscataway, New York, United States) and incubated with antibody with rocking overnight at 4 °C. Protein G Sepharose was added and samples were incubated for 1 h at 4 °C with rocking. Samples were washed three times for 5 min each in lysis buffer, and the Protein G Sepharose-antibody-antigen pellet was resuspended in Laemmli buffer and boiled for 10 min. Samples were run on SDS-PAGE and transferred to nitrocellulose membrane (Schleicher and Schuell Bioscience, Keene, New Hampshire, United States). Western blot signals were developed using the enhanced chemiluminescence kit from AmershamPrimary keratinocytes were culture in low-calcium medium as previously described . TransieSnail promoter was generated by PCR using a forward primer with an XbaI linker sequence, 5′-TCTAGAATTGTTTGCTGCTGTATGGTCTTC-3′, along with a reverse primer with a BglII linker sequence, 5′-AGATCTGTTGGCCAGAGCGACCTAG-GTAG-3′, and mouse genomic DNA as a template. The PCR product was purified with the Gel Extraction Kit  and ligated into pCRII-TOPO TA vector . The promoter was verified by sequencing and digested with XbaI and BglII and subcloned into the pβ-gal BASIC vector . The point mutations in the SMAD binding element was generated with the Quik-Change Kit  using the forward primer 5′-GGGCGGGCTTAGGTGTTTTCATTTACTCTTGAGGAAAAGCTTGGC-3′ and the reverse primer 5′-GCTTTT-CCTCAAGAGTAAATGAAAACACCTAAGCCCGCCCTGCCC-3′. The probes for the Snail in situ hybridization were generated against the 3′ UTR by PCR using the forward primer 5′-ACCTTCTCCCGCATGTCCTTGCTCC-3′ and the reverse primer 5′-CTGCTGAGGCATGGTTACAGCTGG-3′, and genomic DNA as a template. The PCR product was gel purified and ligated into pCRII-TOPO TA vector. The pre-LIM domain of Ajuba was generated essentially as described [The 2.2-kb murine escribed , but wasSnail was used as a template to generate digoxigenin-labeled sense and antisense riboprobes (Roche). The respective probes were obtained by XhoI and BamH1 digestions. In situ hybridizations were performed on 10-μm thick sections of E17.5 mouse embryos. The sections were fixed with 4% PFA for 10 min at room temperature, prehybridized at room temperature for 4.5 h, hybridized with the probe (2 μg/ml) at 55 °C for 12–14 h, blocked with 10% NGS, and treated with anti-dig Fab-AP antibody (Roche #1093274) at a 1:2,500 dilution for 3 h. The sections were incubated with NBT and BCIP until adequate signal was detected.The pCRII-TOPO TA vector containing a region of the 3′ UTR of Tissue samples for immunofluorescence were frozen in OCT and sectioned 10 μm thick on a cryostat. Sections were fixed in 4% paraformaldehyde for 10 min at room temperature, blocked, and stained with antibodies. Tissue samples for immunohistochemistry were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Samples were sectioned on a microtome (10 μm thick) and rehydrated prior to staining with antibody. Samples stained with Snail, pMAPK, pSmad2, and cyclin D were antigen unmasked with 10 mM sodium citrate (pH 6) in an Antigen Retriever 2100 (Pickcell Laboratories, Leiden, Netherlands). The DAB substrate kit (Vector Labs) was used according to manufacturer's instructions to develop the signal.Snail forward: 5′-CAGCTGGCCAGGCTCTCGGT-3′; Snail reverse: 5′-GCGAGGGCCTCCGGAGCA-3′; GAPDH forward 5′-CGTAGACAAAATGGTGAAGGTCGG-3′; and GAPDH reverse: 5′-AAGCAGTTGGTGGTGCAGGATG-3′.RNA was extracted from keratinocytes or skin tissue with Trizol (Invitrogen) according to the manufacturer's protocol. cDNA was generated using oligo-dT primers and the Superscript II kit (Invitrogen). The primers used for PCR were


Cytology slides are often unique and irreplaceable. Unlike surgical pathology cases, where additional paraffin sections can be cut, cytology slides often cannot be duplicated because there are only a few direct smears or the diagnostic material is present on a single slide. Cytology slides are often "sent out" to other physicians, laboratories or hospitals, typically so that the pathologist at the institution where the patient will receive treatment can review the slides. Less often, a cytology lab sends out the slides for a second opinion or as part of the discovery process in a lawsuit, where they may or may not be defendants. Rarely, unique and irreplaceable cytology slides are lost. This article presents a hypothetical scenario that is based on reported state appellate court decisions. The article discusses some of the legal issues that will affect the defendant cytologist/cytology lab and the "expert cytologist," and suggests some steps a cytologist/cytology lab can take to minimize the risk of repercussions from a lost unique and irreplaceable cytology slide. A WestLaw search  uncovered only a handful of reported appellate cases that directly applied to the issue of lost cytology slides. There are many cases and statutes dealing with lost and altered evidence (other than cytology slides), but the circumstances surrounding cytology slides are unique. I did not see any previous reviews or commentary specifically addressing the topic. Notwithstanding the relative paucity of on-point legal authority, the issue is one commonly addressed by cytology laboratories and cytologists and many have approached the issue thoughtfully and prudently.This review synthesizes the available legal cases and presents to practicing cytologists a short, relatively concise summary in the format of a hypothetical case. The review seeks to incorporate the legal issues with the practical aspects of running a cytology laboratory. The review answers how some state courts might approach the problem of lost and irreplaceable cytology slides, and offers general ideas to cytologist for minimizing the risk from lost slides.The issues surrounding the decision whether to send out diagnostic patient slides are more important in cytology than in surgical pathology because the cytology slides are typically unique and irreplaceable. Unlike surgical pathology cases, where additional paraffin sections can be cut, cytology slides often cannot be duplicated because there are only a few direct smears or the diagnostic material is present on a single slide.Slides are routinely "sent out" for a variety of reasons. Most commonly, slides are sent to another institution because the patient's pathology slides will be reviewed before treatment. Some smaller laboratories with only one or two pathologist may send out slides as part of their quality assurance procedures. More rarely, but increasingly, slide's are requested as part of existing or contemplated litigation. Slides might be lost in any of these circumstances. A laboratory or hospital might loose the slides and not discover their absence until the slides are requested.This article will discuss the approach the US legal system takes in addressing what happens when cytology slides are lost, and what steps a prudent laboratory might take to manage the risk. The article also intends to improve cytopathologists' awareness and understanding of some of the legal issues that arise when evidence is missing and perhaps promote an international comparative discussion of alternative legal approaches.In the US, most of medical malpractice law is made and interpreted by state legislatures and state courts. What follows is a hypothetical story based on several legal decisions made by appellate state courts in the United States and reported in the legal literature. Although based on real cases and available to the public, the names have been changed. Importantly, none of this is intended as legal advice and readers should consult their attorney about specific questions.Rugged Labs (RL) is a small, independent laboratory. Part of Rugged Labs' work involves providing Big Giant Lab (BGL) with overflow services for cytopathology. In December of 1995, BGL bought out RL.In 1994 and 1995, a RL cytotechnician interpreted two Pap smears from 30-year-old Ms. Penny as normal. In 1996 a cervical biopsy from Penny showed adenocarcinoma. Ms. Penny underwent a radical hysterectomy, which confirmed the invasive endocervical adenocarcinoma. Ms. Penny is alive today, but endured extended post-operative hospitalization.At the time of the hysterectomy, Penny Plaintiff's oncologist requested that the 1994 and 1995 PAP smear slides be sent to Dr. Experta, who interpreted both Pap smears as containing "abnormal cell groups consistent with adenocarcinoma." Dr. Experta also reviewed the biopsy and in a note concluded that the cells on the Pap smears were consistent with the adenocarcinoma diagnosed on the cervical biopsy.Approximately 6 months later, Plaintiff Penny decided to sue for medical malpractice based on failure to diagnose her endocervical adenocarcinoma on the Pap smears.At some time before the plaintiff filed her lawsuit Dr. Experta's assistant apparently mailed the slides back to BGL. The Pap smear slides are lost, presumably in the mail, by BGL or Dr. Experta's office.RL, the independent laboratory, asked the trial court to grant it summary judgment on the basis that the evidence was lost and no questions of fact remained. Summary judgment means that there are no outstanding questions of fact and the court needs to decide only questions of law. Summary judgment means there is no trial with a jury or judge hearing and weighing evidence. The question of law RL wanted the court to determine on summary judgment was that the lost slides substantially prejudiced RL and summary judgment was, therefore, appropriate. RL included in its motion for summary judgment an affidavit from an expert stating that she could not give an opinion without having the slides to look at. An affidavit by Dr. Experta's secretary stated that the slides were returned to BGL by US mail. BGL submitted an affidavit attesting they did not lose the slides. The parties to the lawsuit stipulated that the slides were lost. The trial court agreed with RL that there should be summary judgment in RL's favor, reasoning that no questions of fact needed to be answered and that a trial would unduly prejudice RL because of the spoliation of evidence. For a summary of the facts, please see Fig. 1.The Plaintiff appealed and the state appellate court reversed the trial court, concluding that the trial court made a mistake in not allowing the case to go to trial. The appeals court reasoned that the case turned on a question of fact; "The slide either showed the presence of cancer cells or it did not." The appellate court envisioned the trial as follows: the cytotechnologist from RL would "testify to her conclusions" and the plaintiff would have Dr. Experta testify to her conclusions. There should be a trial because there were questions of fact including, in the words of the majority, whether the slide had "cancer cells" or not.The majority also discounted the affidavit from BGL, stating that the conclusory statement that BGL had not lost the slides was not valid; just because BGL couldn't find the slides did not mean they never had them. In a footnote, the majority noted that although they aren't accusing anyone, they couldn't help but notice that missing the slides benefited BGL and RL.One judge dissented in the three-judge panel that decided the case. The dissent focused on two points. First, the dissent emphasized that the defendant RL had nothing to do with loosing the slides. Although the plaintiff, Ms. Penny, had no "direct role" in the loss of evidence, the dissent treated Dr. Experta as an agent of the plaintiff and concluded that Dr Experta should have sent the slides back to RL not GBL. The dissent also reasoned that its approach would "encourage experts to treat more carefully evidence delivered into their hands."Secondly, the dissent emphasized that the defendant RL is disadvantaged by the loss of the slide and the plaintiff has gained a significant advantage. The dissent sees a trial where the cytotechnologist's faces a serious credibility problem because her testimony will come across as blatantly self-serving. The cytotech will be limited to "opining that he made no error." Moreover, the fact-finder may conclude that the cytotechnologist is wrong since many may presume that the expert pathologist must be right. Finally, the defendants cannot obtain their own expert to bolster their case, because no cytology slides remain for review.The dissent concludes that summary judgment for RL was proper because the trial court had good reasons to conclude that the lost evidence was going to unduly prejudice the defendant RL.The most obvious issue this case brings up is the difficulty in deciding what to do when it isn't clear who lost the evidence, or when a third party lost the evidence. In contrast, is the situation where one party is responsible for inadvertently losing the evidence or, even worse, where one party deliberately loses or destroys evidence. This is called spoliation of evidence and includes meaningful alteration of the evidence.The court will determine the severity of the sanction for spoliation by the degree of willfulness or bad faith and the extent of the prejudice suffered by the non-responsible party. For example, in one case, surgeons performed a hepatectomy after a small needle core liver biopsy was interpreted as cancer. The hepatectomy specimen showed only cirrhosis and no cancer was found. The small needle liver biopsy was subsequently lost by the hospital. The patient sued for medical malpractice, claiming that the core biopsy was misdiagnosed and lead to an unnecessary hepatectomy. The trial court instructed the jurors that they could "draw the strongest possible inference against [the hospital] as to what the lost cytology slides would have shown." In other words, losing the slide means your opposition can make the slide show whatever they want. Interestingly, in the hepatectomy case, the defendants prevailed by arguing that the diagnosis of cancer and the decision to undergo resection were reasonable, regardless of the cytology results, because the patient had active hepatitis B and a suspicious liver mass on imaging. They argued that even with a negative cytology result the surgeons would have gone ahead with the hepatectomy.But our case is different. Neither party, according to the majority, is responsible for losing the slides, or put another way, either party might be responsible for losing the slides. Several judicial options exist and none are neutral. One is to impose no sanctions and to proceed as usual, only without the slides, as the majority opinion advocated. This likely favors the plaintiff, particularly when there is a subsequent surgical specimen with cancer. A second option is to try to determine which party the court thinks is more prejudiced by the lost slides and then impose a legal remedy, as the trial court did in our hypothetical case by granting summary judgment. The difficulty is that it will not be clear which side is more prejudiced until the slide is recovered. The critical question of whether the negative diagnosis fell below the standard of care can likely be adequately answered only if the parties and their experts can review the slide. A third option might be to not allow Dr. Experta's testimony if the defendant can show that the expert knew or should have known that there was going to be litigation . ArguablInterestingly, each option leads to a different result based on bias about which party is ultimately more responsible or more likely responsible for loosing the slides and a bias about the standard of care. The majority's opinion included a note that the benefit to BGL from losing the slides can't be ignored, a clear statement that the plaintiff had no responsibility for losing the slides, and a simplistic view about the standard of care reflected by the statement that "the slide either showed the cancer cells or it did not." The majority's subtext is that the plaintiff did nothing wrong and they weren't completely sure about the laboratory. The dissenting opinion, in contrast, treated Dr. Experta as the plaintiff's agent and was dissatisfied that Dr. Experta returned the slide to BGL instead of RL, even though at the time BGL had purchased RL. The dissent also showed a more nuanced understanding of the standard of care, conveying skepticism about Dr. Experta's look back conclusion that the Pap smears were "consistent" with adenocarcinoma. The dissent reasoned that perhaps the cells are consistent with malignancy only in the retroscope and that a defense expert might reasonably conclude that it was "not below the standard of care to determine the biopsy negative" were the slides available for review.Penny vs. GBL, a court does not dismiss the plaintiff's case, the absence of the cytology slide will likely impact the defendant cytologist more adversely because many people will assume that the cancerous cells were on the slide and the cytologist or cytotechnologist missed the cancer, which, after all, was present on the subsequent biopsy. In the hypothetical's facts, this was particularly true since Dr. Experta had already opined that the Paps were "consistent with adenocarcinoma." The defendant cytologist/cytology lab is at a serious disadvantage if the court allows Dr. Experta's opinion as admissible evidence. The majority's comment that GBL benefited from the lost slides and the appellate court's decision to allow the plaintiff to go to trial, suggests the court's bias that they believed Dr Experta's interpretation was the correct one.The facts often surrounding a cytopathology medical malpractice case are that there is a subsequent biopsy or surgical specimen with a discrepant diagnosis. If, as in the hypothetical Admittedly, a cytology lab or cytologist does benefit if slides are lost and the court does not attach any responsibility for losing the slides to the potential laboratory or cytologist defendant, because the plaintiff will not have enough evidence to prevail. Similarly, if the slides are discarded after the legal time periods the likelihood of a successful lawsuit is slim because the plaintiff will not have enough evidence, and the defendant complied with legal requirements regarding slide retention. The hypothetical case of Penny vs GBL differs. Remember that once the court allows the case to go to trial, GBL only benefits from absent slides if the defendant initially misinterpreted the slides. If the slides contained no malignant cells, and Dr. Experta over-interpreted the slides, then GBL is prejudiced by not being able to show the slide.Cytology slides are typically in possession the cytology labs. The slides may be sent out for a variety of reasons, but at least initially the lab has possession of the cytology slides. This is both an advantage and a disadvantage for the lab. The disadvantage is that if slides are lost while in the lab's possession a court will typically see it as the lab's responsibility to safeguard the slides. This allows the plaintiff to have the jury infer whatever is best for the plaintiff's case; that the slide had malignant cells when the cytology diagnosis was benign or that there were only benign cells when the diagnosis was malignant, as in the hepatectomy case.The advantage for the cytology lab is that it is in a position of relative control. The lab can implement a system to help reduce the chances of losing a slide and reduce the risk if a slide is lost. Although the lab may want to employ the help of an attorney experienced in these matters, there are several steps every cytology lab can take. First, the lab should ensure that cytology slides are retained for the time that the current federal CLIA regulations, applicable state regulations and the CAP checklist require. All glass cytology slides must be retained for at least 5 years and fine needle aspiration slides retained for 10 years. (Some state regulations may require longer times.) The CAP checklist also requires policies for "protecting and preserving the integrity and retrieval of original slides in cytopathology" and "to ensure defined handling and documentation of the use, circulation, referral, transfer and receipt of original slides to ensure availability of materials for consultation and legal proceedings." Keeping careful records about when and which slides are released to whom is essential for reducing the risk of losing slides. In the send out cases where slides are sent out for a routine second opinion not sought in contemplation of a law suit or because a patient will be treated elsewhere, the lab might obtain the borrower's explicit written agreement that the borrower has responsibility for the slides with an explicit provision about a duty to indemnify the cytology lab for any losses due to a lost slide or slides. A documented telephone call to request the return of tardy slides may also be worthwhile. In cases where slides are requested in contemplation of a lawsuit, the lab may be able to implement a policy that review of slides is done at the lab, ensuring that the lab retains possession. Alternatively, the lab may pursue or agree to a court order requiring production of the slides that clearly addresses who is responsible for the slides and includes an indemnity clause in the event the slides are lost.It is reasonable to make a distinction between sending out non-reproducible slides to an institution that will treat the patient and sending out non-reproducible slides to the plaintiff's expert witness. It is, therefore, important that the lab understands the purpose for which the slides are requested. A documented telephone conversation may clarify the purpose of the outside review and allow the lab to appropriately "triage" the case.Laboratory administrators should understand that the cytology lab serves a public function in safeguarding cytology slides. Court's have recognized a public policy reason to have the laboratory safeguard slides, in part so that the slides are available in the event of malpractice litigation. At the same time, many states have statutes that give patients the right to examine and copy their medical records . These two propositions are not mutually exclusive. One state case considered the question of whether a patient/plaintiff had a right to "immediate possession of pathology slides" and concluded that the plaintiff did not. The court decided that the patient's rights did not exceed the patient's statutory right in the slides. In other words the court was not going to find a common law, or customary, right in the slides that gave the patient a greater right than the applicable statute. The decision noted that the legislative history of the statute included remarks that pathology slides were part of the medical record. The judges then approached the second question about what to do when the "medical record" cannot be duplicated, as with a Pap smear or other cytology slide. In answering this question, the court noted that hospitals and laboratories have public as well as private duties. One of their public duties is to retain slides so that the slides are available in the event of malpractice litigation. This meant that patients do not have a legal right to possess parts of the medical record that cannot be duplicated. The court, however, did not grant the lab complete authority to never release the slides. Since the public policy reason depended fundamentally on preserving slides to help the legal system run smoothly, the decision reminded the laboratory or hospital that, pursuant to the clear terms of a statute, it must send the original slides to a "licensed institution, laboratory or physician" at the patient's written request.Moore vs Regents of the University of California [The dissent characterized the pivotal issue differently and concluded that the patient had a right to immediate possession because the slides contained the patient's cells and she had the right "to control one's body." The dissent reasoned that recent advancement in genetic science and the accompanying difficult privacy issues raised by genetic information strengthened the patient's right to possession of the cells on the glass slide. The majority addressed this argument and, citing the well known case of lifornia , remindePenny v GBL illustrates, lost slides can result in problems for cytologists and the cytopathology laboratory A good place to start is to ensure that existing CLIA regulations, applicable state regulations and other guidelines, such as the CAP checklist, are in place in the laboratory. Thinking about the issue and implementing appropriate risk management strategies with the help of an attorney are also prudent measures.In summary, lost slides can be a problem for cytology laboratories and cytologists, whether responsible for losing the slides or not. The prudent cytologist will minimize the risk of lost slides, because, as the hypothetical case of


Arabidopsis thaliana, with a high number of duplicated genes, as cross-hybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific.Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Microarray platforms based on either oligonucleotides or purified amplification products have been utilised in parallel to produce large amounts of data. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags (ESTs) is, however, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. The use of the arrays is exemplified by analysis of gene expression changes caused by a four-hour indole-3-acetic (auxin) treatment. A total of 270 genes were identified as differentially expressed (120 up-regulated and 150 down-regulated), including several previously known auxin-affected genes, but also several previously uncharacterised genes.We present an alternative solid-phase purification strategy suitable for efficient preparation of short, biotinylated and highly specific probes suitable for large-scale expression profiling. Twenty-one thousand Arabidopsis transcripts.The described solid-phase procedure can be used to prepare gene sequence tag microarrays based on short and specific amplified probes, facilitating the analysis of more than 21 000  Arabidopsis thaliana has been limited when compared to other model organisms, such as human and mouse, mainly due to the lack of high-quality cDNA microarrays offering genome-wide coverage. However, during the recent years both academic and commercial alternatives to these cDNA arrays have emerged. The public initiative by the CATMA consortium has aimed at the production of high-quality probes for each of the 29 787 genes predicted in the Arabidopsis genome [Arabidopsis ESTs compared with 6 million for the human species). Commercial alternatives for genome-wide monitoring of the Arabidopsis transcriptome have been developed by Affymetrix, Agilent Technologies, MWG Biotech, Operon and others. In a recent study the CATMA, Affymetrix and Agilent arrays were found to perform equally, but with a minor advantage for the CATMA arrays in terms of dynamic range [Extensive transcriptional profiling of the plant model system s genome ,2. The ds genome ,3,4, sinic range .Arabidopsis plants treated with physiological concentrations of the well-known plant hormone indole-3-acetic acid (auxin). Finally, we compare our results with those obtained in two previous studies [In the first phase of the CATMA program 21 120 GSTs covering more than 70% of the predicted genes were designed. The length of the probes is kept low to ensure specificity and ranges from 150 bp to 500 bp, with the size distribution heavily shifted towards the shorter fragments. To further increase the specificity of the GSTs their distribution is shifted towards the 3'-end of the genes, with 60%, 16% and 24% representing 3'-, centre and 5'-regions, respectively [ studies  carried Arabidopsis thaliana [In this study we present a method suitable for purification of gene sequence tags, which have recently been designed and successfully used for transcriptional profiling of the plant model system thaliana ,3. The pthaliana . We exemThe amplification products we use to study the purification process are 500 bp, 1 kb and 1.3 kb in length, covering the size range typically used for probes on cDNA arrays. For successful purification of the probe both efficient capture by the beads and release is important. An example of the capture of a 50-μl PCR product and subsequent release is shown in Figure Repeated use of the magnetic beads after a single round of capture and elution is the key feature of the described strategy. To investigate the cross-contamination between iterative cycles of purification as well as the total number of bead purifications that can be used without significant loss in performance, we used agarose gel electrophoresis, DNA Lab-on-chip technology as well as a more sensitive approach based on printing of eluates onto glass slides and hybridisation using a fluorescently labelled oligonucleotide complementary to the purified and printed probes. A carry-over free purification requires that all captured product is released at the first elution so that no product is transferred to the next sample to be purified using the same set of beads. We analysed the presence of cross-contamination by analysing the eluates of two consecutive release reactions from a single immobilised product . Furthermore, the cross-contamination issue was analysed by using the reversible beads in six sequential capture reactions containing either a PCR product or a water-only control in an alternating order . A released product is detected in the first eluate, as expected, but not in the second, by all three methods including the sensitive fluorescence assay. As shown in Figure We studied the capacity of the beads after multiple capture and release cycles by using a constant amount of the three PCR products as input for each iterative purification cycle. An extra washing step was carried out between the purification cycles. Data for nine consecutive binding, washing, elution and regeneration rounds of the PCR products is presented in Figure Arabidopsis biotinylated gene sequence tags (GSTs), with sizes ranging from 150 to 500 bp. The use of GSTs in transcript profiling offers improved specificity when compared to the more common EST or cDNA libraries since each GST has been designed to have minimal cross-hybridisation to other genes, including members of the same gene family. The investigated set of GSTs covers approximately 70% of the genes in the genome, as described in more detail by the CATMA consortium [We applied the described method for purification of 21 120 Arabidopsis thaliana seedlings. Total RNA is collected at three post-treatment time points and compared, using a reference design, to RNA extracted from untreated plants. A general overview of the data is shown in Figure Aux/IAA genes. The Aux/IAA genes encode small short-lived nuclear proteins that interact with the ARF (auxin response factor) family of transcription factors and are thought to modulate the transcriptional activity of the ARFs in an auxin-dependent manner. These ARFs have been shown to bind to auxin-responsive elements (AuxREs) that are found in promotors of several auxin-regulated genes [IAA5 and IAA19 being strongly up-regulated (>30- and 10-fold, respectively) already at 30 minutes, while a two-hour treatment is required for the IAA7 transcripts to reach a two-fold up-regulation. In a recent independent study where Arabidopsis seedlings were treated for only 15 min with 1 μM IAA, all of the Aux/IAA genes listed in IAA7, were found to be up-regulated [Aux/IAA genes four members of the GH3 family that have also been shown to be induced by auxin exhibited a rapid and sustained 2- to 8-fold up-regulation in our study, again confirming the findings reported in a previous study on auxin regulation of gene expression using Affymetrix 8 k oligonucleotide arrays [PIN1 [PIN7 are up-regulated by auxin whereas in contrast the expression of several members of the Aquaporin gene family [The arrays generated by large-scale purification of GSTs are used in a pilot time-point study where the plant hormone indole-3-acetic acid (IAA), also known as auxin, is used to cause transcriptional changes in ed genes . The ARFed genes , and theed genes ,19. Thirys [PIN1  and PIN7e family  are downArabidopsis gene sequence tags for use as microarray probes in transcriptional profiling. Biotinylated amplification products are rapidly captured and eluted using a reusable streptavidin-coated solid-phase support in an automated high-throughput manner directly compatible with subsequent microarray printing. Our results demonstrate that the assembly and purification of gene-specific tags is an alternative to currently used purification methods, especially suitable for short amplification products such as gene sequence tags. In addition, the possibility to generate single-strand probes in the range of 150–500 nucleotides by a sodium hydroxide treatment of immobilised probes with subsequent elution of the remaining biotinylated strand, opens up for new microarray applications that would extend probe length beyond current oligonucleotide synthesis limits.We have described an efficient procedure for large-scale purification of gene sequence tags that can be used for several purposes including microarray fabrication. We demonstrate the utility of the technology by applying it to generate more than 21 000 short (150 – 500 bp) and highly specific The performance of the described purification method was investigated by varying either the amount of beads, the length of the incubation time for binding of the biotinylated product to the streptavidin-coated beads and the number of times the beads were reused. We also investigated if multiple reuses of the beads did introduce a well-to-well cross-contamination. The section below describes the general aspects of the purification and is followed by a more detailed description of the experiments carried out to investigate the different above-mentioned aspects of the purification method.2, 200 nM dNTPs (Amersham Biosciences Europe GmbH, Sweden), 5 pmole universal sequencing primer (USP, 5'-TAAGCTAGGCACTGGCCGTCGTTTTACAACG-3', MWG Biotech AG, Germany), 5 pmole biotinylated reverse sequencing primer (RSP, 5'-AGGCCTAATGGTCATAGCTGTTTCCTGTGTG-3', MWG Biotech AG) and 1.5 units Platinum Taq DNA Polymerase (Invitrogen AB, Sweden). The temperature cycling (5 min at 95°C, 30 × (30 s at 95°C, 30 s at 64°C, 2 min at 72°C), 10 min at 72°C) was carried out in a Hybaid thermal cycler (Thermo Electron Molecular Biology, MA, USA). Pooling and splitting into aliquots of 50 μl in a clone-wise manner was used to remove variances introduced by the amplification step.For all experiments three randomly chosen EST clones (0.5 kb, 1 kb and 1.3 kb) were amplified in 50-μl reactions containing 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2O. By use of a peltier thermal element, the immobilised products kept in suspension were heated in deionised water to 80°C (1°C / 2 s) for 1 second and cooled to room temperature (1°C / 2 s). Efficient elution is achieved through a combination of elevated temperature, appropriate temperature ramping and incubation at the elevated temperature, as described in more detail elsewhere [All purification steps, including bead dispensing, binding of the biotinylated product to the streptavidin moieties, washing, elution and regeneration of beads, were carried out in the Magnatrix 1200 automated workstation (Magnetic Biosolutions AB, Sweden). The biotinylated amplification products were bound to Dynabeads M-270 Streptavidin beads  during an incubation at room temperature using a high-salt binding buffer [1 M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5% PEG-6000 and 0.1% Tween-20] and when bound, washed with 1 × TE-buffer [10 mM Tris-HCl, pH 7.5, 1 mM EDTA]. During incubation the beads were kept in suspension by mixing through pipetting every third minute. Elution was achieved by breaking the streptavidin-biotin bond in a 20-μl volume using deionised HFor all the purifications described below an aliquot of the pooled PCR product corresponding to a 50-μl reaction was used. The binding capacity of the beads was studied using an increasing amount (10 μg, 50 μg, 100 μg and 150 μg) of fresh beads (first use) while keeping the incubation time constant at 20 minutes. To estimate the variability of the method eight independent purifications were carried out (n = 8). The effect of the length of the incubation time was studied using 150 μg of beads and eight different incubation times (1, 5, 10, 15, 20 or 30 min) with four replicates of each (n = 4). To study the effects of multiple reuses of the beads, the same beads were reconditioned and used up to nine times. The amount of beads in the first capture was 150 μg and the capture time 20 minutes. The variability was estimated using four independent replications. The multiple reuse of the beads was also analysed using a hybridisation based approach. Twelve clones ranging from 0.3 to 2 kb were amplified and purified multiple times using reconditioned beads. The purified products were subsequently printed in eight replicates onto glass slides and quantified using Syto61 (Molecular Probes Inc, OR, USA). The well-to-well carry-over of product was analysed by first purifying one of the PCR products, followed by a purification reaction with no PCR product added (water-only control). This pattern was repeated six times for all three products, while the same set of beads was used for all purifications. Eluates from all these purifications were printed on slides and hybridised with a Cy5-labelled oligonucleotide complementary to the common vector sequence present in all products. Hybridisation was carried out using 10 pmole of the labelled oligonucleotide for 1 hour at 35°C in a hybridisation solution containing 50% formamide, 5 × SSC and 0.1% SDS. Slides were washed with 2 × SSC containing 0.1% SDS (5 min at room temperature) and three times with 1 × SSC (1 min at room temperature). Scanning using the G2565BA DNA microarray scanner (Agilent Technologies) was carried out at the highest possible photo multiplier tube setting in order to reveal low-level signals.2, 200 nM dNTPs, 20 pmole of each primer (forward primer biotinylated, Thermo Hybaid GmbH, Germany) and 1.5 units Platinum Taq DNA Polymerase in a total volume of 50 μl. Biotinylated products from each amplification reaction were bound to 200 μg of Dynabeads M-270 Streptavidin beads (reused up to six times) during a 15-minute incubation at room temperature using a high ionic-strength buffer, washed with 1 × TE-buffer and, finally, eluted with 12 μl deionised water. After each capture round the beads were reconstituted, pooled plate-wise and randomly assigned to a new plate. An equal volume of 99.9% dimethyl sulfoxide (DMSO) (Sigma-Aldrich Sweden AB, Sweden) was added and the purified amplification products were arrayed into 22 by 22 patterns in 48 individual blocks with the QArray arrayer (Genetix Limited, UK) and SMP2.5 pins . When dried, printed products were immobilised to the reactive surface of the Ultra-GAPS slides (Corning B.V. Life Sciences, The Netherlands) using 250 mJ/cm2 UV-light .Initial amplification from BAC-clones or genomic DNA was carried out by the CATMA consortium at different nodes throughout Europe ,2. One pArabidopsis Col-0 seedlings were grown at 22°C in MS medium (Duchefa AB, The Netherlands) supplemented with 0.5% sucrose and using a 24-h photoperiod with 16 h of light at 75 mE m-2 sec-1 PAR. The samples were treated with 1 μM indole-acetic acid for a period of 0, 30, 120 and 240 minutes, washed once with an excess of MS medium with 0.5% sucrose for 5 minutes, frozen in liquid nitrogen and stored at -70°C. For each time point, frozen seedlings from three independent vials were pooled, grinded and total RNA extracted using the RNeasy kit (Qiagen GmbH, Germany). The quality of the RNA was determined using the RNA 6000 Nano kit and the Bioanalyzer instrument (Agilent Technologies, CA, USA).10-day-old 20VN, MWG Biotech AG) was annealed to 20 μg total RNA after a denaturating step (10 min at 70°C). The cDNA synthesis reaction was carried out at 42°C for 1 h 45 min in a 30-μl reaction containing 2 mM dNTPs , first-strand buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2), 0.01 mM DTT and 400 units Superscript II (Invitrogen AB). The synthesis reaction was terminated by addition of EDTA, the RNA strand hydrolysed with NaOH (15 minutes at 70°C) and the reaction neutralised with HCl . The cDNA strands were purified using the MinElute spin columns (Qiagen GmbH) with the provided wash and elution buffers replaced by 80% ethanol and 100 mM NaHCO3, pH 9.0, respectively. Monofunctional NHS-ester Cy3 or Cy5 fluorophores (Amersham Biosciences) were coupled to the amino-allyl groups during a 90-minute incubation at room temperature after which unincorporated ester groups were inactivated through a hydroxylamine treatment . The pooled labelling reactions were purified using MinElute spin columns and hybridised to the arrays using a two-step protocol in the GeneTac hybridisation station (Genomic solutions Ltd, UK). The pre-hybridisation at 42°C for 45 min (5 × SSC, 1% BSA (Sigma-Aldrich), 0.1% SDS, 40 μg poly(dA) (Sigma-Aldrich) and 20 μg tRNA (Sigma-Aldrich)) was followed by a 16–18 h hybridisation at 42°C with the labelled material and a hybridisation buffer containing 5 × SSC, 25% formamide, 0.1% SDS, 40 μg poly(dA) and 20 μg tRNA. The slides were washed with 2 × SSC and 0.1% SDS at 42°C, followed by 0.1 × SSC + 0.1% SDS at room temperature and finally by three repeated washes with 0.1 × SSC at room temperature. Slides were scanned at 10-μm resolution using the G2565BA DNA microarray scanner for which the photo multiplier tube (pmt) setting was adjusted so that the images for the Cy3 and Cy5 channels were in balance as determined by visual observation. Each time point-to-reference sample comparison was carried out on two arrays, with the dye labels exchanged between the replicated hybridisations in order to avoid sequence-dependent labelling and hybridisation effects. A control self-to-self hybridisation was also carried out for the untreated sample in order to assess the level of noise in the experimental system.Ten μg anchored oligo dT primer (dTThe acquired tiff-images were processed using the GenePix 4.1 software (Axon instruments Inc, CA, USA) and the data with the R environment for statistical computing , BiocondCATMA a complete Arabidopsis thaliana transcriptome microarrayEST expressed sequence tagGST gene sequence tagIAA indole-3-acetic acidMIAME minimum information about a microarray experimentVW carried out the laboratory work and the data analysis and participated in the design of the study and drafting of the manuscript. AH and ML participated in the solid-phase purification procedure. PN participated in the array production. PH designed and provided the CATMA probes. MU participated in the automation of the solid-phase procedure. RB carried out the auxin treatment and participated in the interpretation of the expression data. JL conceived of the study, participated in the drafting of the manuscript and coordinated the study. All authors read and approved the final manuscript.List of genes identified as up-regulated by the indole-3-acetic acid treatment. Group labels refer to (A) previously identified auxin regulated, (B) transcription related, (C) signal transduction related, (D) transport, (E) cell wall establishment related, (F) metabolic enyzmes, (G) light related, (H) disease repsonse related, (I) other and (J) unknown genes.Click here for fileList of genes identified as down-regulated by the indole-3-acetic acid treatment. Group labels refer to (A) previously identified auxin regulated, (B) transcription related, (C) signal transduction related, (D) transport, (E) cell wall establishment related, (F) metabolic enyzmes, (G) light related, (H) disease response related, (I) other and (J) unknown genes.Click here for file


Although, the transgenic animal mutation assay is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation, the results for the majority of substances were in agreement. Both test systems, the transgenic mouse assay and the mouse bone marrow micronucleus test, have advantages and they complement each other. However, the transgenic animal assay has some distinct advantages over the micronucleus test: it is not restricted to one target organ and detects systemic as well as local mutagenic effects.As part of a larger literature study on transgenic animals in mutagenicity testing, test results from the transgenic mutagenicity assays ( This and the following presentation are part of a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in chemical toxicity testing and mechanistic research. It was decided to compare the results obtained from those transgenic mutagenicity test systems where considerable data was available, with other in vivo genotoxicity tests: in the first article (Part 1) with the mouse bone marrow micronucleus test and in the second (Part II) with the mouse spot test.in vivo mammalian test system for the detection of chromosomal aberrations [The mouse bone marrow micronucleus test is one of several available rrations -5. A docrrations . This telacI model [® mouse), and the lacZ model [Mutagenicity assays using transgenic animals have been developed in particular the cI model  (commerccZ model  (commercMicronuclei are chromatin-containing bodies in the cytoplasm arising from acentric chromosome fragments or from whole chromosomes that were not incorporated in the daughter nuclei during the last stages of mitosis. Chromosome fragments are associated with the clastogenic (chromosome breakage) activity of the test substance whereas the presence of a whole chromosome is indicative of an adverse effect of the test substance on the mitotic spindle apparatus (aneugenic effects). The difference in size of the micronucleus is therefore an indicator for clastogenicity  or aneugenicity (large micronucleus). However, the size of the micronucleus is an inaccurate measure. Micronuclei can be distinguished by further criteria, for example by identification of the presence of a kinetochore or centromeric DNA, indicating aneugenic activity. Overall, an increase in micronuclei is an indirect measure of induced structural or numerical chromosome aberrations ,2.In the micronucleus test according to the OECD guideline 474, erythroblasts in the bone marrow of mice (or rats) are used as target cells. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded. Any micronucleus that has been formed may remain in the otherwise anucleated cytoplasm and can easily been detected. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage .in vivo systems for testing chromosomal aberrations (e.g. the cytogenetic test).In the last three decades, toxicologists have often used the mouse bone marrow micronucleus test because 1) it is part of the regulatory toxicology in the admission procedure for chemicals and drugs and 2) it has advantages in speed, simplicity, and cost effectiveness in comparison to other target gene that serves as target for mutations, and a shuttle vector for recovering the target gene DNA from the tissue of the transgenic animal. The transgene is constructed using recombinant DNA technologies.Transgenic mutation test systems contain a foreign gene construct having two essential parts: the transgene containing the ® mutagenesis assay system contains about 30–40 copies of a lambda LIZα shuttle phage vector integrated into its genome at a single locus on chromosome 4. The target site for mutagenesis is the lacI gene [The proprietary mouse of the Big BlueacI gene .lacI gene form blue plaques, whereas bacteria with a nonmutated lacI form colourless plaques. The ratio between blue and white plaques is a measure of the mutagenicity. [The test compound is administered to the mouse either as a single or repeated dose. After the post treatment period for manifestation of the DNA lesions, the tissue of interest is isolated and the DNA extracted. A proprietary lambda DNA packaging extract automatically excises the lambda vector target and packages it into a lambda phage head and the phage is transfected to bacteria. These bacteria are plated on agar indicator plates containing the chromogenic substance . The phage transfected bacteria with mutations in the enicity. ,9.lacZ shuttle vector for the lacZ mouse model contains the entire lacZ target gene [lacZ mouse model contains about 80 copies of the shuttle vector at chromosome 3.The lambda-gt10-get gene . The comlacZ- E. coli strain (for transfection) in the presence of the chromogenic substance  as indicator. The plaques containing an intact lacZ are β-galactosidase active and are blue, whereas plaques containing mutated lacZ will be white/colourless. In this original model the ratio between colourless and blue plaques is a measure of the mutagenicity. Due to optical difficulties in the evaluation of plaques, this system has been improved using a selection assay in lieu of colour screening whereby only mutant particles form plaques [or mutant lacZ gene). The ratio between the number of plaques produced under selective conditions versus the number of plaques under non-selective conditions is a measure of the mutagenicity [As above, the test compound is administered to the mouse, the genomic DNA is isolated from the tissue of interest, and the lambda genomes are excised by and packed with a bacteriophage packaging extract. The resulting phage particles are then plated on a  plaques . The numgenicity ,10.Data presented in this documentation are the results of an extensive literature research. Concerning data on transgenic mouse assays only primary literature was used. Data on the mouse bone marrow micronucleus assay were extracted from reliable reviews on this item or from primary literature. For all other data informations from secondary literature or data compilation bases were used.The authors are aware that a comparison of the transgenic mouse assays with the mouse bone marrow micronucleus test is limited by the fact that different genotoxic endpoints are studied in these two systems. In transgenic mouse assays, point mutations and small insertions and deletions are detected whereas in the mouse bone marrow assay, chromosome breakage leading to light microscopically visible micronuclei resulting from chromosome fragment or micronuclei originated from whole chromosomes are investigated. However, both point mutations and micronuclei may be induced by a single agent, so some overlap of results is to be expected.and the Muta™mouse assay (n = 29) or the Big Blue® mouse assay (n = 21) or both transgenic mutation assays (n = 11); see ® mouse assay, the results obtained with 14 out of 21 substances agreed with results in the mouse micronucleus test, but 6 showed no agreement and another was inconclusive.From the literature, 39 substances were identified with data on the mouse bone marrow micronucleus test Most substances included in this comparison are also carcinogenic in long-term assays on mice. No data on carcinogenicity in mice are available for 4-acetylaminofluorene and negative results were obtained with bromomethane and 2,6-diaminotoluene.in vitro and in vivo (see The following 17 substances were gene mutagenic in at least one of the examined organs in the Muta™mouse assay, induced micronuclei in mouse bone marrow and showed positive results in carcinogenicity studies on mice: 2-acetylaminofluorene, 4-aminobiphenyl, benzo(a)pyrene, 1,3-butadiene, chlorambucil, cyclophosphamide, 7,12-dimethylbenz(a)anthracene, ethylmethanesulfonate, N-ethyl-N-nitrosourea, methylmethane-sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, 4-nitroquinoline-1-oxide, N-nitrosodimethylamine, procarbazine, quinoline, and urethane. Further studies on chromosome aberration vivo see  supporte® mice (see in vivo studies on clastogenic effects in bone marrow of rats and mice.Of the substances tested for mutagenic activity in bone marrow in Muta™mice, 14 gave positive results, only 1,3-butadiene, methylmethanesulfonate, and quinoline were negative, indicating that other organs are more sensitive. 1,3-Butadiene is clearly clastogenic in other studies on chromosome aberration in mice and induced gene mutation in the bone marrow of Big Bluemice see . Quinoli® mouse assay, chromosome mutagenic effects in the mouse bone marrow micronucleus assay and induced carcinogenic effects in long-term assays on mice,: 2-acetylaminofluorene, aflatoxin B1, benzene, benzo(a)pyrene, 1,3-butadiene, cyclophosphamide, 7,12-dimethylbenz(a)anthracene, ethylene oxide, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, N-nitrosodimethylamine, urethane. In the Big Blue® mouse assay the target organ bone marrow revealed also increased mutation frequencies induced by benzene, 1,3-butadiene, and 7,12-dimethylbenz(a)anthracene. However, negative results in bone marrow were obtained with cyclophosphamide, ethylene oxide, and N-nitrosodimethylamine, indicating that other target organs are more sensitive in the Big Blue® mouse assay. All of these 12 substances induced also chromosome aberrations in majority of further in vitro and in vivo studies.12 substances showed gene mutagenic effects in at least one of the examined organs in the Big Bluein vivo tests on the endpoint chromosome aberration including a cytogenetic test on mice [The inconclusive result in the bone marrow micronucleus test  is contr on mice ,19,21. M on mice . Overallin vitro studies on chromosome aberrations but IQ induced micronuclei in rats (see IQ is mutagenic in the liver of the Muta™mouse  but negarats see . This di® assay on mice mutagenic activity was detected in the bladder but not in the liver [The target organ of carcinogenesis in mice (and humans) is the bladder . In the he liver . As expehe liver ,36 gave ® mice after inhalation [Local carcinogenic effects were observed in carcinogenicity studies, the lung is the target organ after inhalation -40. Mutahalation . As expe® mouse assays examining the liver [in vivo are inconclusive  . Positivhe liver -84. The sive see .in vivo genotoxicity endpoints have shown almost negative results after single exposure but genotoxic activity after repeated application, for example in the mouse bone marrow micronucleus assay [in vivo as well as in vitro test systems revealed gene mutagenic effects.This substance induced no mutagenic effects in lung, liver, or bone marrow of the Muta™mouse which were target organs in mouse carcinogenicity studies ,120. Theus assay . Positiv® mouse [in vitro and in vivo assays clearly supported the clastogenic activity [Only weak mutagenic effects in the liver but no effects in bone marrow were observed in the Muta™mouse ,124 and ® mouse -113,126.® mouse ; other iactivity ,122. Theactivity  have shoin vivo and in vitro assays on the endpoint chromosome aberration revealed clearly positive results [A very similar situation is given with mitomycin C. No mutagenic activity was observed in the Muta™mouse assay in liver and bone marrow after single injection but the same dose induced chromosome aberrations in the bone marrow of the same mice . The mou results -141.in vivo studies gave no clear indication for chromosome aberration  and in bone marrow (rat) -163. Sevable see .® mouse, one gave negative [in vitro) or negative results were obtained in other studies on clastogenicity or other endpoints of genotoxicity  reason for the negative results in the Muta™mouse assay might be that the MTD was not reached [This substance gave clearly positive results in different carcinogenicity studies on mice . Althoug reached . Overall® mouse assay, mutagenic activity was detected in the kidney in gavage studies[This might be a further example for a substance where genotoxic effects are not related to the target organ bone marrow but induce systemic effects in others. The target organ in oral carcinogenicity studies on mice and rats was the kidney, in mice systemic carcinogenic effects were also seen in lung and liver -190. In e studies,191. Thee studies as well e studies-190.in vitro mammalian test systems indicated gene mutagenic activity [This substance showed mutagenic activity in the Muta™mouse assay  but incoactivity  supportiin vivo tests are equivocal phthalate  whose mutagenic (and carcinogenic) potential could not be demonstrated with the mouse bone marrow micronucleus test but with the transgenic mouse assay. This might be due to the fact that the micronucleus test is restricted to the bone marrow as target organ. In contrast, all organs can be examined in transgenic mouse assay for mutagenic activity without any restrictions.A special subgroup of substances should also be mentioned: substances with predominantly local mutagenic/carcinogenic effects and less systemic direction of effects. For these substances the mouse bone marrow micronucleus test is an unsuitable test system.N-nitrosodi-N-propylamine has shown positive results in different target organs including the bone marrow using the transgenic mouse assay but negative results were shown in the mouse micronucleus test.Beside the restrictions on the target organ there is also given the possibility that a substance induces predominantly gene mutations and not or less chromosome aberrations at the same dose level. The example in vivo. These genotoxic effects are not easily detectable with the transgenic mouse assay which is restricted to the detection of small deletion and insertions in the DNA. However, the negative Muta™mouse assay on mitomycin C has some limitations in the validity of the test system. With increased dose levels (MTD reached) and/or repeated application also gene mutagenic effects might be detected in the transgenic mouse assay.On the other hand there are also substances for which the transgenic mouse assay is an unsuitable test system. The examples methylmethanesulfonate and mitomycin C have shown that chromosome aberration and not gene mutation is the predominant endpoint at the corresponding dose level Interestingly, substances with carcinogenic effects induced by nongenotoxic mechanisms gave mainly correct negative results in both test systems although the protocols for the transgenic mouse assays were not optimised except for chloroform.® mouse assay combined, and the mouse bone marrow micronucleus test are documented in Table and mutagenic effects in transgenic mice as well as mutagenic effects in the mouse bone marrow micronucleus test  some differences were apparent between the two test systems.The overall accuracy of the micronucleus test is lower than that of the transgenic mouse assays. This is mainly due to 11 negative results in the micronucleus test system (negative in the micronucleus test but positive in carcinogenicity studies) influencing the terms sensitivity and negative predictability. Three of these negative results in the micronucleus test are obtained with carcinogenic substances [chloroform, di-(2-ethylhexyl)phthalate, and tetrachloromethane] for which carcinogenic effects are considered to be of a nongenotoxic mechanism. However, chloroform, di-(2-ethylhexyl)phthalate and tetrachloromethane gave negative results in transgenic mice, so the comparison of both test system is not essentially affected and the evaluation „nongenotoxic“ supported. For the other 8 substances out of these 11 with false negative results in the micronucleus test these results are explainable (see above): Ortho-anisidine (mutagenic/carcinogenic effects are restricted to the bladder), IQ (bone marrow presumably not target organ of genotoxicity in mice and more gene mutagenic than clastogenic), asbestos , 2,4-diaminotoluene (target organ liver, presumably not bone marrow), N-nitrosodiethylamine (target organ liver, more gene mutagenic than clastogenic), N-nitroso-N-propylamine (presumably more gene mutagenic than clastogenic), beta-propiolactone , tris(2,3-dibromopropyl)phosphate (systemic effects not related to the bone marrow).The term negative predictability is also low in the transgenic mouse assay due to false negative results on six carcinogenic substances; for three of them, hydrazine, mitomycin C, and tetrachloroethylene (detailed treatise in section „direct comparison“, see above) genotoxic mechanisms are presumed. For hydrazine (no repeated application) and tetrachloroethylene (MTD not reached) limitations on the experimentel design might be the reason for the negative results. Mitomycin C is clearly more clastogenic than gene mutagenic, and the transgenic mouse with lacI and lacZ is possibly an unsuitable test system. For 3 out of the 6 substances the carcinogenic effects in mice were attributed to nongenotoxic mechanisms: chloroform, di-(2-ethylhexyl)phthalate and tetrachloromethane , all gave negative results in transgenic mice.Only two substances with negative results in long-term carcinogenicity studies are available in the data pool: bromomethane and 2,6-diaminotoluene. Both gave correct negative results in the transgenic mouse assay  but false positive results in the micronucleus test (see term specificity).Generally, the differences between the two test systems might be due to the fact that 1) unequal genotoxic endpoints are investigated (chromosome aberration in the micronucleus test versus gene mutation in the transgenic mouse assay), 2) organotrophy of genotoxic effects  might play an essential role and 3) transgenic rodent assay conditions in the different systems may not be optimal for mutation detection.A comparison of the transgenic mouse assays with the mouse bone marrow micronucleus test is limited by the fact that different genotoxic endpoints are studied in these two systems. In transgenic mouse assays, point mutations and small insertions and deletions are detected whereas in the mouse bone marrow assay, chromosome breakage leading to light microscopically visible micronuclei resulting from chromosome fragment or micronuclei originated from whole chromosomes are investigated.In comparison to other test systems in genotoxicity testing using endogenous target structures the spontaneous mutant frequency in the transgenic mouse assay is relatively high. This might be related to the fact that bacterial DNA is the target gene (high methylation rate) or the transgene is silent and no transcription related repair occurs like in endogenous genes which are more efficiently repaired . In the Comparing the target organs and cells at risk at the time of exposure, the mouse micronucleus test is restricted to one target organ, the bone marrow, especially to the erythroblasts. This limitation is not given in transgenic mouse assays: target cells are cells in all organs .Both test systems are similar in the number of animals used for a valid test. The minimal number of mice needed in the mouse bone marrow assay is 25 per gender  using a treatment schedule with 2 or more applications at 24 h intervals and sampling 18–24 h following the final treatment. In the limit test (for a test substance of low toxicity) only one dose level of 2000 mg/kg bw is necessary (OECD guideline 474, ).In transgenic mutation assays ca. 20 animals  are recommended per species and gender ,197. In Due to the simplicity of the mouse bone marrow micronucleus assay and the use of systems for automated analysis, this test is less expensive than the transgenic mouse assay.A comparison both test systems is presented in Table ® mouse and the Muta™mouse were compared with those from the more traditional mouse bone marrow micronucleus test. The transgenic animal mutation assay, which is not yet used in toxicological screening, is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation. However, from the 39 substances, the majority gave the same positive or negative result in both test systems. The substances where differences occurred were discussed in more detail. The advantages and disadvantages of the transgenic Big Blue® mouse and the Muta™Mouse transgenic model compared to the micronucleus test were discussed and both systems were found to have a place in mutagenicity testing and to supplement each other. The transgenic animal assay has, however, some distinct advantages over the micronucleus test in that it is not restricted to one target organ and detects systemic as well as local mutagenic effects.In a comparison of the tests available to genetic toxicologists, the results from studies on substances which had been tested in transgenic mutagenicity assays Big BlueUW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript.Table: Results in the transgenic mouse assay versus mouse bone marrow micronucleus testClick here for file