Temperature-dependent elasticity of DNA, RNA, and hybrid double helices.

Nucleic acid double helices in their DNA, RNA, and DNA-RNA hybrid form play a fundamental role in biology and are main building blocks of artificial nanostructures, but how their properties depend on temperature remains poorly understood. Here, we report thermal dependence of dynamic bending persistence length, twist rigidity, stretch modulus, and twist-stretch coupling for DNA, RNA, and hybrid duplexes between 7°C and 47°C. The results are based on all-atom molecular dynamics simulations using different force field parameterizations. We first demonstrate that unrestrained molecular dynamics can reproduce experimentally known mechanical properties of the duplexes at room temperature. Beyond experimentally known features, we also infer the twist rigidity and twist-stretch coupling of the hybrid duplex. As for the temperature dependence, we found that increasing temperature softens all the duplexes with respect to bending, twisting, and stretching. The relative decrease of the stretch moduli is 0.003-0.004/°C, similar for all the duplex variants despite their very different stretching stiffness, whereas RNA twist stiffness decreases by 0.003/°C, and smaller values are found for the other elastic moduli. The twist-stretch couplings are nearly unaffected by temperature. The stretching, bending, and twisting stiffness all include an important entropic component. Relation of our results to the two-state model of DNA flexibility is discussed. Our work provides temperature-dependent elasticity of nucleic acid duplexes at the microsecond scale relevant for initial stages of protein binding.

Pollinator visitation closely tracks diurnal patterns in pollen release.

PREMISE: Animal-pollinated plants face a high risk of pollen loss during its transfer. To limit the negative effect of pollen losses by pollen consumption and heterospecific transfer, plant species may adjust and stratify their pollen availability during the day (i.e., "schedule" their pollen presentation) and attract pollinators in specific time frames. METHODS: We investigated diurnal patterns of pollen availability and pollinator visitation in three coflowering plant species: Succisa pratensis with open flowers and accessible pollen, pollinated mainly by pollen-feeding hoverflies; Centaurea jacea with open flowers and less accessible pollen, pollinated mainly by pollen-collecting bees; and Trifolium hybridum with closed flowers and pollen accessible only after the active opening of the flower, pollinated exclusively by bees. RESULTS: The three plant species differed in the peak pollen availability, tracked by the visitation activity of their pollinators. Succisa pratensis released all pollen in the morning, while pollinator activity was still low and peaked with a slight delay. In contrast, C. jacea and T. hybridum had distinct pollen presentation schedules, peaking in the early afternoon. The pollinator visitation to both of these species closely matched their pollen availability. CONCLUSIONS: Stratifying pollen availability to pollinators during the day may be one of several mechanisms that allow coflowering plants to share their pollinators and decrease the probability of heterospecific pollen transfer.

Remarks on Geomatics Measurement Methods Focused on Forestry Inventory.

This contribution focuses on a comparison of modern geomatics technologies for the derivation of growth parameters in forest management. The present text summarizes the results of our measurements over the last five years. As a case project, a mountain spruce forest with planned forest logging was selected. In this locality, terrestrial laser scanning (TLS) and terrestrial and drone close-range photogrammetry were experimentally used, as was the use of PLS mobile technology (personal laser scanning) and ALS (aerial laser scanning). Results from the data joining, usability, and economics of all technologies for forest management and ecology were discussed. ALS is expensive for small areas and the results were not suitable for a detailed parameter derivation. The RPAS (remotely piloted aircraft systems, known as "drones") method of data acquisition combines the benefits of close-range and aerial photogrammetry. If the approximate height and number of the trees are known, one can approximately calculate the extracted cubage of wood mass before forest logging. The use of conventional terrestrial close-range photogrammetry and TLS proved to be inappropriate and practically unusable in our case, and also in standard forestry practice after consultation with forestry workers. On the other hand, the use of PLS is very simple and allows you to quickly define ordered parameters and further calculate, for example, the cubic volume of wood stockpiles. The results from our research into forestry show that drones can be used to estimate quantities (wood cubature) and inspect the health status of spruce forests, However, PLS seems, nowadays, to be the best solution in forest management for deriving forest parameters. Our results are mainly oriented to practice and in no way diminish the general research in this area.

RNA kink-turns are highly anisotropic with respect to lateral displacement of the flanking stems.

Kink-turns are highly bent internal loop motifs commonly found in the ribosome and other RNA complexes. They frequently act as binding sites for proteins and mediate tertiary interactions in larger RNA structures. Kink-turns have been a topic of intense research, but their elastic properties in the folded state are still poorly understood. Here we use extensive all-atom molecular dynamics simulations to parameterize a model of kink-turn in which the two flanking helical stems are represented by effective rigid bodies. Time series of the full set of six interhelical coordinates enable us to extract minimum energy shapes and harmonic stiffness constants for kink-turns from different RNA functional classes. The analysis suggests that kink-turns exhibit isotropic bending stiffness but are highly anisotropic with respect to lateral displacement of the stems. The most flexible lateral displacement mode is perpendicular to the plane of the static bend. These results may help understand the structural adaptation and mechanical signal transmission by kink-turns in complex natural and artificial RNA structures.

Polyandrous bee provides extended offspring care biparentally as an alternative to monandry based eusociality.

Parental care behavior evolves to increase the survival of offspring. When offspring care becomes complicated for ecological reasons, cooperation of multiple individuals can be beneficial. There are two types of cooperative care: biparental care and worker (helper)-based care (e.g., eusociality). Although biparental care is common in several groups of vertebrates, it is generally rare in arthropods. Conversely, eusociality is widespread in insects, especially the aculeate Hymenoptera. Here, we present a case of biparental care in bees, in Ceratina nigrolabiata (Apidae, Xylocopinae). Similar to eusocial behavior, biparental care leads to greater brood protection in this species. Male guarding increases provisioning of nests because females are liberated from the tradeoff between provisioning and nest protection. The main benefit of parental care for males should be increased paternity. Interestingly though, we found that paternity of offspring by guard males is extraordinarily low (10% of offspring). Generally, we found that nests were not guarded by the same male for the whole provisioning season, meaning that males arrive to nests as stepfathers. However, we show that long-term guarding performed by a single male does increase paternity. We suggest that the multiple-mating strategy of these bees increased the amount of time for interactions between the sexes, and this longer period of potential interaction supported the origin of biparental care. Eusociality based on monandry was thought to be the main type of extended brood protection in bees. We show that biparental care based on polyandry provides an interesting evolutionary alternative.

Complex approach to skin repair in an extensively burned child: a case study.

The limited amount of donor sites and loss of dermis are major challenges in the therapy of extensively burned patients. Here, we present a complex treatment approach of an eight-year-old boy with full-thickness burns on 90% of the total body surface area, using simple and efficient techniques of tissue engineering. To obtain sufficient skin for grafting we repeatedly harvested the same anatomical areas. Acceleration of donor site healing was achieved by treatment with a suspension of noncultured autologous skin cells (NASC) and acellular porcine dermis (Xe-Derma (XD), Czech Republic). Moreover, such wound management allowed up to six reharvestings, compared with one-to-three procedures following routine treatment. Bilayer Integra template (Integra LifeSciences Corp., US) was used as the dermal substitute in over 60% of full-thickness burns. Following successful vascularisation of the neodermis in 3-4 weeks, the templates were covered with meshed split-thickness skin grafts (STSG), or Meek autografts, and facilitated by NASC/XD. We may conclude that such a 'sandwich' technique approach, combining four biological covers (Integra, STSG, NASC and XD), significantly contributed to the successful skin repair of the patient.

Local application of adipose-derived mesenchymal stem cells supports the healing of fistula: prospective randomised study on rat model of fistulising Crohn's disease.

OBJECTIVE: Local application of adipose-derived mesenchymal stem cells (ADSC) represents a novel approach for the management of perianal fistula in patients with Crohn's disease. A randomised study on an animal model was performed to investigate the efficacy and to detect the distribution of implanted ADSCs by bioluminescence (BLI). MATERIALS AND METHODS: A caecostomy was used as a fistula model in 32 Lewis rats. The ADSCs were isolated from transgenic donor expressing firefly luciferase. Animals were randomly assigned to groups given injections of 4 × 106 cells (n = 16, group A) or placebo (n = 16, group B) in the perifistular tissue. Fistula drainage assessment was used to evaluate the fistula healing. After application of D-luciferin, cell viability and distribution was detected using an IVIS Lumina XR camera on days 0, 2, 7, 14 and 30. RESULTS: The fistula was identified as healed in 6 (38%) animals in group A vs. 1 case (6.3%) in group B (p = .033). The BLI was strongest immediately after administration of ADSCs 31.2 × 104 (6.09-111 × 104) p/s/cm2/sr. The fastest decrease was observed within the first 2 days when values fell by 50.2%. The BLI 30 days after injection was significantly higher in animals with healed fistulas - 8.23 × 104 (1.18-16.9 × 104) vs. 1.74 × 104 (0.156-6.88 × 104); p = .0393. CONCLUSIONS: Local application of ADSCs resulted in significantly higher fistula closure rate on an animal model. BLI monitoring was proved to be feasible and showed rapid reduction of the ADSC mass after application. More viable cells were detected in animals with healed fistula at the end of the follow-up.

5-fluorouracil Toxicity Mechanism Determination in Human Keratinocytes: in vitro Study on HaCaT Cell Line.

5-fluorouracil (5-FU) and capecitabine therapy is often accompanied by palmar-plantar erythrodysesthesia (PPE) which is manifestation of 5-FU toxicity in keratinocytes. The main mechanisms of 5-FU action are thymidylate synthase (TS) inhibition which can be abrogated by thymidine and strengthened by calciumfolinate (CF) and incorporation of fluorouridinetriphosphate into RNA which can be abrogated by uridine. For proper PPE treatment 5-FU mechanism of action in keratinocytes needs to be elucidated. We used the 5-FU toxicity modulators uridine, thymidine and CF to discover the mechanism of 5-FU action in human keratinocyte cell line HaCaT. To measure the cellular viability, we used MTT test and RTCA test. CF did not augment 5-FU toxicity and 5-FU toxicity was weakened by uridine. Therefore, the primary mechanism of 5-FU toxicity in keratinocytes is 5-FU incorporation into RNA. The uridine protective effect cannot fully develop in the presence of CF. Thymidine addition to 5-FU and uridine treated cells not only prevents the toxicity-augmenting CF effect but it also prolongs the 5-FU treated cells survival in comparison to uridine only. Therefore, it can be assumed that in the presence of uridine the 5-FU toxicity mechanism is switched from RNA incorporation to TS inhibition. Although particular 5-FU toxicity mechanisms were previously described in various cell types, this is the first time when various combinations of pyrimidine nucleosides and CF were used for 5-FU toxicity mechanism elucidation in human keratinocytes. We suggest that for PPE treatment ointment containing uridine and thymidine should be further clinically tested.

Mesenchymal stem cells seeded on cross-linked and noncross-linked acellular porcine dermal scaffolds for long-term full-thickness hernia repair in a small animal model.

Biological meshes are biomaterials consisting of extracellular matrix that are used in surgery particularly for hernia treatment, thoracic wall reconstruction, or silicone implant-based breast reconstruction. We hypothesized that combination of extracellular matrices with autologous mesenchymal stem cells used for hernia repair would result in increased vascularization and increased strength of incorporation. We cultured autologous adipose-derived stem cells harvested from the inguinal region of Wistar rats on cross-linked and noncross-linked porcine extracellular matrices. In 24 Wistar rats, a standardized 2×4 cm fascial defect was created and repaired with either cross-linked or noncross-linked grafts enriched with stem cells. Non-MSC-enriched grafts were used as controls. The rats were sacrificed at 3 months of age. The specimens were examined for the strength of incorporation, vascularization, cell invasion, foreign body reaction, and capsule formation. Both materials showed cellular ingrowth and neovascularization. Comparison of both tested groups with the controls showed no significant differences in the capsule thickness, foreign body reaction, cellularization, or vascularization. The strength of incorporation of the stem cell-enriched cross-linked extracellular matrix specimens was higher than in acellular specimens, but this result was statistically nonsignificant. In the noncross-linked extracellular matrix, the strength of incorporation was significantly higher in the stem cell group than in the acellular group. Seeding of biological meshes with stem cells does not significantly contribute to their increased vascularization. In cross-linked materials, it does not ensure increased strength of incorporation, in contrast to noncross-linked materials. Owing to the fact that isolation and seeding of stem cells is a very complex procedure, we do not see sufficient benefits for its use in the clinical setting.

Temperature-Dependent Twist of Double-Stranded RNA Probed by Magnetic Tweezer Experiments and Molecular Dynamics Simulations.

RNA plays critical roles in the transmission and regulation of genetic information and is increasingly used in biomedical and biotechnological applications. Functional RNAs contain extended double-stranded regions, and the structure of double-stranded RNA (dsRNA) has been revealed at high resolution. However, the dependence of the properties of the RNA double helix on environmental effects, notably temperature, is still poorly understood. Here, we use single-molecule magnetic tweezer measurements to determine the dependence of the dsRNA twist on temperature. We find that dsRNA unwinds with increasing temperature, even more than DNA, with ΔTwRNA = -14.4 ± 0.7°/(°C·kbp), compared to ΔTwDNA = -11.0 ± 1.2°/(°C·kbp). All-atom molecular dynamics (MD) simulations using a range of nucleic acid force fields, ion parameters, and water models correctly predict that dsRNA unwinds with rising temperature but significantly underestimate the magnitude of the effect. These MD data, together with additional MD simulations involving DNA and DNA-RNA hybrid duplexes, reveal a linear correlation between the twist temperature decrease and the helical rise, in line with DNA but at variance with RNA experimental data. We speculate that this discrepancy might be caused by some unknown bias in the RNA force fields tested or by as yet undiscovered transient alternative structures in the RNA duplex. Our results provide a baseline to model more complex RNA assemblies and to test and develop new parametrizations for RNA simulations. They may also inspire physical models of the temperature-dependent dsRNA structure.

Human keratinocyte growth and differentiation on acellular porcine dermal matrix in relation to wound healing potential.

A number of implantable biomaterials derived from animal tissues are now used in modern surgery. Xe-Derma is a dry, sterile, acellular porcine dermis. It has a remarkable healing effect on burns and other wounds. Our hypothesis was that the natural biological structure of Xe-Derma plays an important role in keratinocyte proliferation and formation of epidermal architecture in vitro as well as in vivo. The bioactivity of Xe-Derma was studied by a cell culture assay. We analyzed growth and differentiation of human keratinocytes cultured in vitro on Xe-Derma, and we compared the results with formation of neoepidermis in the deep dermal wounds treated with Xe-Derma. Keratinocytes cultured on Xe-Derma submerged in the culture medium achieved confluence in 7-10 days. After lifting the cultures to the air-liquid interface, the keratinocytes were stratified and differentiated within one week, forming an epidermis with basal, spinous, granular, and stratum corneum layers. Immunohistochemical detection of high-molecular weight cytokeratins (HMW CKs), CD29, p63, and involucrin confirmed the similarity of organization and differentiation of the cultured epidermal cells to the normal epidermis. The results suggest that the firm natural structure of Xe-Derma stimulates proliferation and differentiation of human primary keratinocytes and by this way improves wound healing.

Creating a Material Spectral Library for Plaster and Mortar Material Determination.

Historic object analysis and the knowledge of composition play an important role in restoration processes. Based on this information, restoration works are conducted. This paper introduces a non-invasive technique of plaster and mortar material decomposition using reflectance spectroscopy. For this purpose, a NIRQuest512-2,5 from Ocean Optics®/Ocean Insight®, is used to create a unique spectral library consisting of various materials. They were carefully selected to include those that were and still are commonly used for a plaster and mortar production. Each material of the spectral library was mapped in detail, verified using scanning electronic microscope (SEM) data, and the results were compared to a previously determined spectral signature. The new spectral library was then tested on 11 unknown plaster and mortar samples and verified using a scanning electronic microscope. It was found that reflectance spectroscopy provides a powerful tool for plaster and mortar material decomposition, although at the moment it cannot fully replace invasive techniques like chemical analyses or other invasive techniques. It provides relevant information that can be used for restoration works.

Nesting of Ceratina nigrolabiata, a biparental bee.

Biparental care is very rare in insects, and it was well-documented in only one bee species to this date - Ceratina nigrolabiata. However, biparental care was only recently discovered in this species, and detailed description of natural history of this species is missing. Here, we describe the nesting cycle of C. nigrolabiata. Pairs of C. nigrolabiata are established before female starts offspring provisioning. After provisioning is finished (when youngest offspring reached larval stage), the male abandons the nest. Males which are present in nests where female already finished provisioning brood cells, are probably mainly temporary visitors. The female can perform long-time offspring guarding, but only 22% of completely provisioned nests are guarded by a female. Most nests (54%) are closed and abandoned, when provisioning is completed, and other (24%) are orphaned before provisioning is finished. Guarded nests have statistically higher number of brood cells provisioned than unguarded nests. Generally, C. nigrolabiata is unique among bees due to its biparental behavior, but it has also uncommon traits of nesting biology among Ceratina bees, e.g. fast offspring development in comparison with provisioning rate, and high proportion of nests which are closed and abandoned by mother.

Phenotyping breast cancer cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis and affinity chromatography for glutathione-binding proteins.

BACKGROUND: Transformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs) and other proteins interacting with glutathione (GSH) in model cell lines could be of particular interest. METHODS: We compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE). We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots. RESULTS: Correlation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics. CONCLUSIONS: Our results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines.

Impact of the Nucleosome Histone Core on the Structure and Dynamics of DNA-Containing Pyrimidine-Pyrimidone (6-4) Photoproduct.

The pyrimidine-pyrimidone (6-4) photoproduct (64-PP) is an important photoinduced DNA lesion constituting a mutational signature for melanoma. The structural impact of 64-PP on DNA complexed with histones affects the lesion mutagenicity and repair but remains poorly understood. Here we investigate the conformational dynamics of DNA-containing 64-PP within the nucleosome core particle by atomic-resolution molecular dynamics simulations and multiscale data analysis. We demonstrate that the histone core exerts important mechanical restraints that largely decrease global DNA structural fluctuations. However, the local DNA flexibility at the damaged site is enhanced due to imperfect structural adaptation to restraints imposed by the histone core. If 64-PP faces the histone core and is therefore not directly accessible by the repair protein, the complementary strand facing the solvent is deformed and exhibits higher flexibility than the corresponding strand in a naked, undamaged DNA. This may serve as an initial recognition signal for repair. Our simulations also pinpoint the structural role of proximal residues from the truncated histone tails.

Two-dimensional electrophoretic comparison of metastatic and non-metastatic human breast tumors using in vitro cultured epithelial cells derived from the cancer tissues.

BACKGROUND: Breast carcinomas represent a heterogeneous group of tumors diverse in behavior, outcome, and response to therapy. Identification of proteins resembling the tumor biology can improve the diagnosis, prediction, treatment selection, and targeting of therapy. Since the beginning of the post-genomic era, the focus of molecular biology gradually moved from genomes to proteins and proteomes and to their functionality. Proteomics can potentially capture dynamic changes in protein expression integrating both genetic and epigenetic influences. METHODS: We prepared primary cultures of epithelial cells from 23 breast cancer tissue samples and performed comparative proteomic analysis. Seven patients developed distant metastases within three-year follow-up. These samples were included into a metastase-positive group, the others formed a metastase-negative group. Two-dimensional electrophoretical (2-DE) gels in pH range 4-7 were prepared. Spot densities in 2-DE protein maps were subjected to statistical analyses (R/maanova package) and data-mining analysis (GUHA). For identification of proteins in selected spots, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed. RESULTS: Three protein spots were significantly altered between the metastatic and non-metastatic groups. The correlations were proven at the 0.05 significance level. Nucleophosmin was increased in the group with metastases. The levels of 2,3-trans-enoyl-CoA isomerase and glutathione peroxidase 1 were decreased. CONCLUSION: We have performed an extensive proteomic study of mammary epithelial cells from breast cancer patients. We have found differentially expressed proteins between the samples from metastase-positive and metastase-negative patient groups.

A simple non-destructive test of cellular activity (NTCA) for in vitro assessment of cancer cell chemosensitivity/resistance.

Determination of chemosensitivity/chemoresistance is becoming increasingly important for individualization of breast cancer chemotherapy. We developed a simple non-destructive test of cellular activity (NTCA) for assessment of the cytopathic effect of antitumour drugs in vitro. Contrary to routinely used methods (e.g. MTT), besides the comparative evaluation of metabolic activity using pH (given by the medium colour), the NTCA enables the simultaneous assessment of proliferation and morphology of cultured cells (phase-contrast microscopy) at any time during the incubation with cytostatics. Moreover, the regenerative potential of the cells can be examined by cell recovery and growth after drug removal. We provide evidence for the relevance of NTCA in chemosensitivity testing of primary breast cancer cells and breast cancer cell lines for cisplatin, gemcitabine and tamoxifen. NTCA represents a simple addition to the chemosensitivity assessment and could also serve for rapid screening of new antitumour strategies.

Arising podosomal structures are associated with neoplastic cell morphological phenotype induced by the microenvironment.

Increased numbers of rosettes of podosomes were observed in overgrown rat Rous sarcoma RsK4 cells. A possible role of these structures in nutrient uptake in tumour cell survival was investigated by exposure to acute starvation. A single cell suspension of RsK4 cells in Hanks balanced salt solution was allowed to interact with either clean uncoated or serum-coated for bait coverglasses. Confocal microscopy revealed contrasting 3D cell morphologies that were associated with conspicuous patterns of podosomal structures, which on the coated coverglasses resembled the sealing zones of osteoclasts, while on the uncoated coverglasses they resembled the marginal podosomes of migrating monocyte-derived cells. Thus, the arising podosomal structures, the involvement of which in an uptake of nutrients appeared feasible morphologically, were associated with the emerging 3D cell shapes guided by the microenvironment. Such phenotypic plasticity of neoplastic RsK4 cells in response to microenvironmental challenge suggested that uniqueness in cellular attributes within the neoplastic cell population could be crucial for the malignant potential.

Human allogeneic keratinocytes cultured on acellular xenodermis: the use in healing of burns and other skin defects.

The tissue engineered skin should be composed of both dermal and epidermal layers. We combined cultured human allogeneic keratinocytes with acellular xenodermis prepared from pig xenografts. The resulting composite skin was termed recombined human/pig skin (RHPS), and could be cultured in both, undifferentiated and differentiated phenotype. The undifferentiated RHPS was grown submerged and formed 1-2 layers of keratinocytes. The differentiated phenotype (D-RHPS) was grown at the air-liquid interface and formed 5-20 cell layers similar to the normal epidermis, including the granular and horny layers. Undifferentiated RHPS has skin-like consistency and has been successfully used for treatment of burns and skin defects using "upside-down" application. Donor sites and deep dermal burn wounds prepared by tangential excision or deep dermabrasion grafted with RHPS healed in the course of about one week after keratinocyte transplantation. Simple acellular xenodermis without keratinocytes can also be used as temporary cover for donor sites, small to medium leg ulcers and other skin defects. Xenodermis can be fully sterilized and stored at the room temperature.

Origin of cells cultured in vitro from human breast carcinomas traced by cyclin D1 and HER2/neu FISH signal numbers.

BACKGROUND: We have developed and optimized a feeder layer method for cultivation of normal human mammary luminal cells and found it suitable for establishing more than 150 primary cultures from individual human breast carcinomas. Here, we investigated if the malignant cells that are in situ additionally characterized by increased numbers of proto-oncogenes can be traced by the FISH method in the ex vivo-derived cultures. MATERIALS AND METHODS: Paraffin sections from 9 tumors with derived cell cultures kept frozen in our cell bank were screened by FISH for cyclin D1 (CCND1) and c-erb-B2 (HER2/neu) proto-oncogene signal numbers. RESULTS: In 6 tumors (5 primary tumors, 1 cutaneous metastasis), increased numbers of FISH signals were found in 55-99% of cells. Then, the relevant cell cultures were FISH screened; in cell populations maintained for up to 2-6 passages in vitro the incidence of cells with increased FISH signals was found to be low (2-16%). Moreover, the cells with multiplied signals that survived more than one passage in vitro were evidently unable to divide further. However, in all 6 tumors at least a small fraction of cells displaying only two signals of CCND1 or HER2/neu genes was identified directly in invasive tumor structures in the vicinity of cells with multiple signals. CONCLUSION: Our findings suggest that these invasive tumor cells displaying only two proto-oncogene signals were most probably involved in the initiation and propagation of ex vivo tumor-derived primary cell cultures.