Fibroblasts prepared from different types of malignant tumors stimulate expression of luminal marker keratin 8 in the EM-G3 breast cancer cell line.

It is widely recognized that stromal fibroblasts significantly influence biological properties of multiple tumors including breast cancer. However, these epithelial-mesenchymal interactions seem to be essential in tumor biology and it is not fully clear whether this interaction is tumor type-specific or has a more general non-specific character. To elucidate this question, we tested the effect of cancer-associated fibroblasts (CAFs) isolated from different types of tumors (breast cancer skin metastasis, cutaneous basal cell carcinoma and melanoma, squamous cell carcinoma arising from oral cavity mucous membrane) on the EM-G3 breast cancer cell line. The results were compared with control experiments using normal human dermal fibroblasts, 3T3 mouse fibroblasts, and 3T3 fibroblasts influenced by the fibroblasts prepared from the basal cell carcinoma. Our results demonstrated that expression of luminal marker keratin 8 was influenced only by CAFs prepared from any tested tumors. In contrast, all tested types of fibroblasts showed a strong stimulatory effect on the expression of basal/myoepithelial marker keratin 14. The CAFs also elevated the number of cells with positivity for both keratins 8 and 14 that are similar to ductal originated precursor cells. The expression of proliferation marker Ki67 was not influenced by any of the tested fibroblasts. In conclusion, our data indicate that CAFs are able to influence the phenotype of a breast cancer cell line and this effect is based on a tumor type-unspecific mechanism. Finally, a clear functional difference between normal and CAFs was demonstrated.

Local treatment of hand-foot syndrome with uridine/thymidine: in vitro appraisal on a human keratinocyte cell line HaCaT.

5-fluorouracil (5-FU) is one of the most commonly used antineoplastic drugs in the anticancer therapy. The hand-foot (HF) syndrome (palmar-plantar erythrodysesthesia) is an adverse effect frequently related to long-term i.v. administration of 5-FU or its orally applicable prodrug capecitabine. Its severity can even lead to interruption of the otherwise effective anticancer therapy. Tentative practice in some clinics has shown that topical application of 10% uridine ointment is beneficial for calming down the HF syndrome. This study is focused on verifying the alleged protective activity of uridine in the in vitro model of cultured human keratinocyte cell line HaCaT. We also tested the protective effects of thymidine alone or uridine-thymidine combination. The cellular viability time progression was measured in order to evaluate the effect of protective agents by three different types of cytopathogenicity tests-NTCA test (non-destructive test of cellular activity), modified MTT test and RTCA (real-time cell analyser, Roche). All three methods proved the ability of uridine and uridine-thymidine combination to protect keratinocytes against 5-FU damage in vitro. While thymidine alone did not show any remarkable effect, the thymidine-uridine combination demonstrated enhanced protective activity compared to uridine alone. Our findings provided the supporting rationale for using uridine or uridine-thymidine ointments in the HF syndrome local therapy.

Reduced inducibility of SOCS3 by interferon gamma associates with higher resistance of human breast cancer lines as compared to normal mammary epithelial cells.

The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the antiproliferative effect of interferon gamma in relation to SOCS3 expression in a panel of breast cancer cell lines and normal mammary epithelial cells. Compared to normal cells most breast cancer lines (7/8) were highly resistant to IFN-gamma. Using Northern blot and real time RT-PCR we investigated transcription of SOCS3 genes. All normal epithelial cells (4/4) showed SOCS3 induction (2-14 fold) while most breast cancer lines did not or weakly activated SOCS3 after the interferon gamma treatment. Among the cancer lines, the MDA-MB-468 cells showed increased sensitivity to IFN-gamma and relatively high level of SOCS3 induction (2-3 fold). Together, there was a good correlation

Thermal, cardiac and adrenergic responses to repeated local cooling.

The aim of this study was to ascertain whether repeated local cooling induces the same or different adaptational responses as repeated whole body cooling. Repeated cooling of the legs (immersion into 12 degrees C water up to the knees for 30 min, 20 times during 4 weeks = local cold adaptation - LCA) attenuated the initial increase in heart rate and blood pressure currently observed in control subjects immersed in cold water up to the knees. After LCA the initial skin temperature decrease tended to be lower, indicating reduced vasoconstriction. Heart rate and systolic blood pressure appeared to be generally lower during rest and during the time course of cooling in LCA humans, when compared to controls. All these changes seem to indicate attenuation of the sympathetic tone. In contrast, the sustained skin temperature in different areas of the body (finger, palm, forearm, thigh, chest) appeared to be generally lower in LCA subjects than in controls (except for temperatures on the forehead). Plasma levels of catecholamines (measured 20 and 40 min after the onset of cooling) were also not influenced by local cold adaptation. Locally cold adapted subjects, when exposed to whole body cold water immersion test, showed no change in the threshold temperature for induction of cold thermogenesis. This indicates that the hypothermic type of cold adaptation, typically occurring after systemic cold adaptation, does not appear after local cold adaptation of the intensity used. It is concluded that in humans the cold adaptation due to repeated local cooling of legs induces different physiological changes than systemic cold adaptation.

Primary intestinal epithelial cells can be infected with laboratory-adapted strain HIV type 1 NDK but not with clinical primary isolates.

Infectivities of HIV-1 primary isolates and laboratory-adapted strains were compared in primary fetal enterocytes and the colonic epithelial cell line HT29. Infection by two laboratory strains, HIV-1 NDK and HIV-1 NDK(A4), which were adapted on CEM and HT29 cells, respectively, produced significant amounts of virus in both target cell systems. Intestinal cells were resistant to infection with HIV-1 primary isolates regardless of their genetic subtype or SI/NSI phenotype. Biological properties of analyzed viruses rather than differences in cultivation system seem to be responsible for differences between these in vitro and ex vivo results.

A recombined skin composed of human keratinocytes cultured on cell-free pig dermis.

Treatment of full skin thickness burns requires replacement of both the dermal and the epidermal components of the skin. We describe a method of preparing recombined human/pig skin (RHPS) by cultivating human keratinocytes on dried cell-free pig dermis (CFPD). CFPD dried on a tissue culture dish forms a thin collagen film which behaves like a firm substrate for cell cultures. HK were grown on the epidermal side of the CFPD using lethally irradiated 3T3 cells as feeders. After reaching confluency of human keratinocytes, human fibroblasts can be cultured on the dermal side of the RHPS. It was possible to obtain approximately 500 cm2 of the RHPS from 1 cm2 human split-skin graft in 3 weeks. RHPS is easy to handle, is similar in structural, mechanical and adhesive properties to the normal skin, and can be meshed. This RHPS might be advantageous for permanent covering of wounds in major burns.

A modification of the gradual fractional trypsinization method for the preparation of rat liver cell cultures.

A new simple technique for the preparation of primary and secondary cultures of rat liver cells is described. Hepatocytes from the liver of 9-day-old rats showed the organotypic morphology, whereas cells from embryonic livers were pleiomorphic.

Metabolic activity of cultured skin grafts cryopreserved in different forms.

Cultured epidermal cells in the form of coherent sheets have been used for the treatment of skin defects. Metabolic activity of fresh and in liquid nitrogen cryopreserved keratinocyte suspensions and three forms of coherent cultured skin sheets were compared with the aim to find the most appropriate form of skin cultures for cryopreservation. Keratinocytes cultured from cryopreserved suspensions formed a confluent layer in 8-10 days after thawing, showing 95% activity of the fresh confluent cultures. Cryopreserved cultured epidermal sheets attached to the bottom of the dish as well as recombined human/pig skin (RHPS) reached more than 70% of the metabolic activity of fresh grafts following 24 h regeneration in the incubator after thawing. Cultured epidermal sheets detached from the dish and mounted on tulle grass reached only 28% of the fresh graft metabolic activity under the same conditions.

Cultivation of psoriatic keratinocytes and an attempt to isolate and detect a specific psoriatic antigen.

Keratinocytes from psoriatic lesion and healthy skin region obtained from the skin of a psoriatic patient were cultivated on lethally irradiated 3T3 cells. They multiplied twice as quickly as normal keratinocytes from healthy skin. Ten antigen fractions were prepared from cell suspensions of psoriatic keratinocytes and from the medium left after their cultivation. Later, they were evaluated in ELISA tests performed with the sera of psoriatic patients and healthy persons. The presence of specific antigens was not demonstrated.

Modified method of in vitro cultivation of human keratinocytes suitable for grafting.

The standard method (Green et al., 1979; Simon and Green, 1985) for cultivating human keratinocytes was simplified and adapted to support growth of large keratinocyte sheets (up to 105 X 280 mm) in glass roller bottles. The keratinocyte sheets can be used to cover burn wounds and skin-graft donor sites.

Quasi-dynamic test of in vitro cell migration developed to characterize dividing cells of spontaneously metastasizing rat sarcomas.

A "Quasi-dynamic Test of in vitro Cell Migration" (QTCM) was developed in which the quantitative estimation of migration of dividing cells is based on a comparative evaluation of their dispersion in single-cell-derived colonies after 72 h of clonal growth under standard and test conditions. Expert visual evaluation of microscopic images of cell colonies including estimation of dispersion of cells in the colonies is checked and made more precise by computerized evaluation of digitized outlines of cells in the colonies. Using QTCM, distinctions in the pattern of migration between three neoplastic cell populations differing by their origin and degree of malignancy were found. Although transfer of cells into slightly acid conditions did not always increase the mean value of cell dispersion in the colonies, each cell population was found to include a cell fraction that did manifest considerably greater migration and this fraction was markedly larger in the more malignant cell populations. The results of both types of evaluation, visual and computerized, were in mutual accord, with accuracy being greater on the computer side. Thus, the QTCM proved useful for qualified estimation and/or computer measurement of in vitro migration of dividing cells on a population level.

Expression of BRCA1, NBR1 and NBR2 genes in human breast cancer cells.

BRCA1 is a tumour suppressor gene with a caretaker function in the DNA-damage repair and the maintenance of genome integrity. The human BRCA1 and NBR2 genes and the homologous Brcal and Nbr1 mouse genes are situated head-to-head on human chromosome 17q21 and on mouse chromosome 11, respectively. Their transcription start sites, located on opposite DNA strands, are separated by 218 bp in humans, and by 289 bp in mice. Because of this intimate contact and because of our previous observation of a quasi-reciprocal expression pattern of Brca1 and Nbr1 in mouse spermatogenesis, we estimated here the relative mRNA expression of BRCA1, NBR1 (next-to-BRCA1) and NBR2 genes in a panel of permanent cell lines and primary cell cultures derived from human breast cancer or normal mammary tissue. The analysis revealed highly significant downregulation of BRCA1 in 11 out of 12 examined tumour cell lines and primary cell cultures as compared to non-malignant mammary cells. Two isoforms of NBR1(1A) and the classical NBR1(1B) transcripts were found in cells from malignant mammary tissues, all of them downregulated in respect to normal cells. The expression of NBR2 differed, being increased in three permanent tumour cell lines and slightly decreased in all primary breast cancer cell cultures. The in silico analysis revealed two new putative domains of the predicted NBR1 protein, suggesting its role in the ubiquitin pathway. The recent identification of the ubiquitin protein ligase activity of BRCA1 implies a possible functional connection between both genes.

Y chromosome and vimentin used to trace the fate of allogeneic keratinocytes delivered to the wound by the recombined human/pig skin.

RHPS, composed of confluent allogeneic keratinocytes cultured on cell-free pig dermis, stimulates wound healing when applied with the keratinocyte layer facing the wound. So far it has not been clarified whether the confluent keratinocytes implanted 'upside-down' can 'take' or only stimulate healing by producing growth factors. Confluent male keratinocytes were grafted onto donor sites of three female patients. Biopsies were taken on days 4, 6 and 9 after grafting. The fate of donor cells was followed in paraffin sections by FISH for the Y chromosome and by persisting expression of vimentin taken as a marker of cultured keratinocytes. Histological evaluation was complemented by detection of keratin 10 and involucrin. All three donor sites healed within one week. On day 4 the early neoepidermis was multilayered but disordered after transplantation. A large proportion of cells were apparently of donor origin as indicated by the presence of Y chromosomes, irregular morphology, expression of vimentin in the bottom and upper layers of the neoepidermis, and by irregular expression of involucrin and keratin 10 only in the central layer of the neoepidermis. From day 6 onwards, the new epidermis acquired an ordered stratification. Involucrin and keratin 10 renewed normal distribution in suprabasal layers. Concomitantly, vimentin expression was decreasing. The Y chromosome was still found on day 6 but not on day 9. We concluded that confluent allogeneic keratinocytes temporarily 'take' to the wound and contribute to rapid wound closure, being replaced by the patient's epidermal cells after about one week.

Necrobiotic process causing burn wound conversion may be prevented by allogeneic keratinocytes delivered by the recombined human/pig skin.

The spontaneous necrobiotic process frequently causes conversion of DDB (deep 2nd degree wounds) into full-thickness skin loss (3rd degree wounds). We found that this process may be positively influenced by the activity of living human allogeneic keratinocytes cultured on acellular pig dermis. This RHPS, if applied 'upside-down' with the epidermal layer facing the wound, provides an opportunity for keratinocytes to influence the healing. The aim of the present study was to find conditions, in terms of timing and wound-bed preparation, for optimum healing activity of RHPS. The wound beds were prepared either with tangential excision, surface dermabrasion or deep dermabrasion. Out of 17 wounds grafted with RHPS after tangential excision, 15 (88%) healed in 4-10 days; early excised wounds (up to day 5) healed within less than 10 days after the injury. Out of 8 wounds grafted after surface dermabrasion, only 2 (25%) healed. Out of 6 wounds grafted with RHPS after deep dermabrasion, 4 (67%) healed. The optimum healing effect of RHPS and prevention of conversion was achieved in early tangentially excised wounds.

Tissue-engineered skin in the treatment of vitiligo lesions.

Vitiligo is characterized by the loss of skin pigmentation due to the destruction of melanocytes. Its treatment is usually difficult. For stable cases, melanocyte transplantation is the method of choice. A newly developed treatment with recombined human/porcine skin methodology, permitting easy handling of the graft, is described in the present work. In five vitiligo patients, autologous epidermal cells were obtained from pigmented thin skin biopsies. The cells were cultured on a dried cell-free porcine dermis by the 3T3 feeder layer technique. After 10 days melanocytes were regularly dispersed in confluent keratinocyte cultures. Upside-down delivery of epidermal cells was used. The epidermal layer was directly applied onto a dermabraded vitiligo lesion, with porcine dermis covering the lesion. Pigmentation started to be visible 4-6 weeks after grafting. After using the above described methodology, the pigmentation appeared in the range of 65-80% of the grafted area. Additional UVA irradiation enhanced the treatment success up to 100%. The surgical vitiligo treatment appears to be a reasonable method of choice in stable vitiligo cases of a disease lasting for at least two years, which means for approximately 5% of all vitiligo patients.

Two dynamic morphotypes of sarcoma cells, asymmetric stellate and triangle with leading lamella, are related to malignancy.

A notion of the dynamic morphotype was developed as a conjunction between cell shape and migration. This enabled the investigation of the relationship between malignancy and patterns of dynamic morphology in neoplastic cells in vitro. Time-lapse cinemicroscopy was used to analyse the cell behaviour of three rat neoplastic cell lines (K2, T15, and A8), differing in metastatic potential, that were instrumental in revealing a coincidence between high migratory activity and appearance of the 3D structure of actin cables in high-malignant A8 cells (Pokorná et al., 1994). A set of criteria was established for visual classification of cell morphology. Matching the pattern of cell morphology with locomotory activity led to identification of four dynamic morphotypes. Cell speed was determined by tracking and the dynamic morphotypes assigned by the operator. All the three cell populations were studied for incidence of the dynamic morphotypes in culture media differing in pH: 6.6 simulating acid extracellular condition in tumours, physiological 7.4, and alkaline 8.2. The results showed that acid pH stimulated motile activity in the intermediate-malignant T15 and most malignant A8 cells. The T15 and A8 cells also manifested a prolonged continuation of fast locomotion in the early G1 phase and displayed a prevalence of two fast moving dynamic morphotypes: asymmetric stellate and triangle with leading lamella.

Clonal expansion of epithelial cells from primary human breast carcinoma with 3T3 feeder layer technique.

3T3 feeder layer technique provided support for clonal growth and serial propagation of two apparently single epithelial cells isolated from a peroperative biopsy of a primary ductal breast carcinoma. The total culture lifetime was estimated to be more than 30 doublings, 21 of which took place during the primary culture. The two cells were the only survivors of two-week exposure to stressing conditions that resembled the microenvironment in a tumour (low pH, depleted nutrition and accumulation of metabolic waste). The epithelial character of the cells was proved by positive immunostaining for keratins 7/17. The majority of growing cells did not express keratin 19. Only quiescent cells in some colonies, which appeared to reach a more advanced stage of differentiation, expressed keratin 19. These features correspond with the characteristics of mammary luminal cells which in vivo undergo differentiation from the stem K19- to secretory K19+ cells. The luminal cells are supposed to be the target of malignant transformation in the mammary gland. The described technique opens a regular way for the in vitro clonal growth of individual primary cells from breast tumours. Such an approach can improve our understanding of the biology of breast cancer cell populations and also simplify the predictive chemosensitivity assay on breast cancer cells from individual patients.

Characterization of the differentiated phenotype of an organotypic model of skin derived from human keratinocytes and dried porcine dermis.

A number of skin models have been developed, but a simple method for rapidly producing large quantities of differentiated epidermis has been missing. We show the differentiated phenotype of human keratinocytes in organotypic culture arising in vitro by air-exposure of keratinocytes cultured with feeders on dried pig dermis. Keratinocytes were seeded at low density on the dermis covered with irradiated NIH-3T3 feeder cells and after reaching confluence lifted to the air-medium interface. A well differentiated epidermis with distinct basal, spinous, granular and stratum corneum layers was formed within 1 week. In this way, 100 cm2 of the differentiated recombined human/pig skin (D-RHPS) can be obtained from 106 secondary keratinocytes in 14 days. The entire keratinocyte life cycle takes place on the dermal substrate - from single cells to stratified epidermis. The differentiation was characterized using a panel of monoclonal antibodies. Similarly as in the normal skin, keratin 14 was expressed in all cell layers, keratin 10 in suprabasal layers, beta1-integrin and epitopes to antibody LH8 in the basal layer, involucrin and transglutaminase in the granular and horny layer of the epidermis. Keratins 16 and 7/17, which are absent in the normal epidermis, but present suprabasally in the psoriatic one, were expressed strongly in all suprabasal layers and in a subpopulation of basal cells. The keratinocytes can be combined with two other cell types cultured either on the dermal side of the dermis and/or on the bottom of the dish. It appears that this simple skin model can be used in studies of epithelial/mesenchymal interactions and interactions between epidermal cells and infectious agents. It may be particularly useful for the study of human papilloma viruses.

Spontaneously metastasizing rat sarcomas LW13K2 and RPS: assessment by the immunogenetic test of malignancy, in vitro behaviour and karyology.

Populations of LW13K2 sarcoma derivatives were compared for their malignancy, patterns of cell behaviour in vitro (dynamic morphology and migration) and karyological pattern. The following tumour cell populations were used: the original LW13K2 sarcoma from inbred LEW/CUB rats, RPS sarcoma derived from it by neoplastic progression, four cell populations isolated in vitro from metastases of a syngeneic LEW-CUB strain rat with RPS tumour and four neoplastic cell populations isolated from spontaneous metastases shed by RPS sarcoma in allogeneic rats, differing from LEW/CUB in weak alloantigenic loci. Although RPS tumour did not grow progressively in MHC-different recipients (while the original LW13K2 tumour did), it grew progressively and metastasized in all groups of non-MHC allogeneic recipients. Parallel with the metastatic potential patterns of in vitro behaviour, such as an increased incidence of the quasi-stellate morphotype at slightly acid culture conditions endowed with enhanced changeability of the cell shape and migrational activity, were found. Cytogenetic analysis demonstrated rather stable chromosomal patterns over the cascade of neoplastic progression from LW13K2 sarcoma over RPS sarcoma to freshly isolated metastases. This indicated that the apparent neoplastic progression observed in the cell populations derived from LW13K2 sarcoma is with high probability not due to the selection at the chromosomal level.

Behaviour of normal and neoplastic rat cells on grooved substrates.

The distribution and shape of rat cells cultivated on plastic surfaces containing cylindrical areas of various radii and grooves of various depths were studied. Normal embryo cells and two lines of neoplastic cells (LW13K2 and RsK4) were used. The nuclear shape, orientation of the nuclei and migration of cells from the bottom of the grooves were assessed by quantitative methods. All characteristics of the behaviour of both neoplastic cell lines on the grooved substrates were found to be different from those of normal cells: a) the nuclei of neoplastic cells, in contrast to those of the normal ones, did not undergo additional elongation on the cylindrical areas of the substrate; b) the orientation of neoplastic cells with regard to the axis of the cylindrical substrate was decreased or absent; c) the migration of neoplastic cells from certain types of the grooves was decreased. It is suggested that the different reaction to the geometry of the substrate may be a characteristic feature of transformed cells. Possible mechanisms of these alterations are discussed.