Cell Kinetic Investigations Research Articles

Optogenetics and Optical Tools in Automated Patch Clamping.

Automated patch clamping (APC) has been used for almost two decades to increase the throughput of electrophysiological measurements, especially in preclinical safety screening of drug compounds. Typically, cells are suctioned onto holes in planar surfaces and a stronger subsequent suction allows access to a whole cell configuration for electrical measurement of ion channel activity. The development of optogenetic tools over a wide range of wavelengths (UV to IR) provides powerful tools for improving spatiotemporal control of in vivo and in vitro experiments and is emerging as a powerful means of investigating cell networks (neuronal), single cell transduction, and subcellular pathways.Combining APC and optogenetic tools paves the way for improved investigation and control of cell kinetics and provides the opportunity for collecting robust data for new and exciting applications and therapeutic areas. Here, we present an APC optogenetics capability on the Qube Opto 384 system including experiments on light activated ion channels and photoactivated ligands.

Significance of cell kinetic parameters in the prognosis of malignant melanoma: a review

Malignant melanoma has been extensively studied concerning methods of predicting progression and clinical outcome. The maximum tumor thickness as measured by Breslow's method is the cornerstone prognostic criterion, but despite this, evolution of the disease in some patients remains unpredictable, confirming that new reliable prognostic factors are awaited. Cell kinetic evaluation has been shown to be a useful tool for assessing the prognosis of breast and gastrointestinal cancer patients. Indeed, in these fields, the mitotic index and MIB-1 expression index, which are indirect estimates of the growth fraction of tumor cell population, are commonly shown to correlate with tumor grade and patient survival and presented as prognostic factors. In melanoma, results of cell kinetic investigations are conflicting: some studies have established a link between high proliferative activity and a bad prognosis, whereas other reports suggest the opposite. The aim of this review is to discuss these findings.

Quantification of hepatocytic proliferation in the laboratory mouse: A comparative study using immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression

The proliferation rate in livers of 120 mice (60 males and 60 females) was analyzed by immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression on ethanol-fixed/paraffin-embedded specimens. Mice were divided into three groups, with 20 males and 20 females in each group: mice in the first group served as controls, while mice in the second and third groups were treated with a low and a high dose, respectively, of a non-genotoxic drug candidate for 2 weeks. A dose-related increase of the proliferating hepatocyte fraction was disclosed by both immunohistochemical methods, reaching statistical significance already in the low-dose male group for BrdU incorporation and in both male and female low-dose groups for PCNA expression. A good correlation between the degree of BrdU and PCNA labeling was observed and, as expected, the percentage of PCNA expressing cells was generally higher than the percentage of BrdU-positive cells. We concluded that the detection of PCNA expression represents a reliable method for the quantification of the hepatocytic proliferating fraction in rodents and allows the use of archival material for cell kinetic investigations in toxicologic pathology.

Cell kinetic investigations in craniopharyngioma: preliminary results and considerations.

In vitro determination of 3H-thymidine labeling index (LI%) was carried out in craniopharyngiomas from 16 patients submitted to repeated surgical procedures. This study showed a poor correlation between LI% values and prognosis, but allowed to detect a high LI% variation in the same tumor investigated in serial different times. The spatial distribution of S-phase proliferative cells in squamous epithelium of cystic components suggests a rationale for intracavitary treatment of cystic and mixed craniopharyngioma and may help explain its success.

Long-term histopathologic evaluation of human expanded skin.

Forty-two patients operated on by skin expansion have been contacted after a mean time of 25 months from the last surgery. Two biopsies have been taken from the expanded area of each patient. In 12 patients it has been possible to obtain a similar sampling from the opposite, nonexpanded area of the body. The samples underwent optic microscopy and cell kinetic and DNA content investigations. The epidermal structure of the followed-up skin, compared with the skin of the opposite side of the body, looks normal. The mitotic activity of the epidermal cells has returned to the values of preexpanded skin. The dermis shows a low degree of elastosis and zonal fragmentation of elastic fibers. The hypodermis, where the expander capsule was removed during the last surgery, does not show an accentuated fibrosis.

Comparison of intradermal injections of ]bromodeoxyuridine and tritiated thymidine in the in vivo measurement of epidermal cell turnover time in goats

Study of keratinisation defects such as seborrhoea may require investigation of epidermal cell kinetics in clinical cases. For this to be practically useful a safe, reliable and quick method is essential. This study compared epidermal cell kinetics in the goat following the use of intradermal injection of tritiated thymidine ([ 3H]TdR) and bromodeoxyuridine ( BURY) to label S phase nuclei. Turnover time for goat epithelial cells was not significantly different for the two agents; 26·3 ± 6·0 days for thymidine and 21·8 ± 5·2 days for burd It was concluded that intradermal injection of BURD will be a suitable technique for investigating epidermal cell kinetics in vivo and could have applications in a clinical situation because it is quicker and safer than [ 3H]TdR.

Cell cycle status of stromal cells in long‐term haematopoietic cultures

This study was performed to further define the mechanism by which the stromal micro-environment regulates haematopoiesis. In long-term marrow cultures the interactions between stromal cells and haematopoietic cells can be investigated at the cellular level. Long-term marrow cultures from hamsters do not require repopulation or addition of hydrocortisone and are suitable for investigation of cell kinetics. The cellular kinetics of haematopoietic and stromal cells, as studied by tritiated thymidine ([3H]dT) incorporation, revealed that DNA synthesis occurred in both the non-adherent and the adherent cells. In established cultures the adherent stromal cells were predominantly in a quiescent non-cycling state: less than 2% adherent cells incorporated [3H]dT within 5 h. Removal of the supernatant cells did not affect the labelling index of adherent cells, since the labelling indices at the 50-75 h time point were 14.3% and 12.5% in the presence and absence of supernatant cells respectively. An apparent stimulus for stromal cells to incorporate [3H]dT was attachment or adhesion. Following replating of supernatant cells of long-term marrow cultures, 23.3% of the reformed adherent layer cells were labelled compared with 12-14% in cultures with previously formed unmobilized adherent cells (P less than 0.01). The data indicate that adherent cells are not required to synthesize DNA for maintenance of haematopoiesis in established long-term marrow cultures, and that recruitment into the cell cycle has an independent mechanism that is not influenced by feed-back from the supernatant cells.

Mitotic rate, DNA distribution, and chromatinin situ sensitivity to heparin in breast cancer

The purpose of this study was to characterize breast carcinomas by cell kinetic parameters. Mitotic rate (MR) and flow cytometrically (FCM) measured cell cycle distribution as well as chromatin testing in situ employing heparin for determination of activated chromatin, provided the following results: MR counted in 73 unselected carcinomas showed an increase up to a tumor size of 4.2 cm (p less than 0.05); beyond this diameter, the MR was found to decrease. In T1-T2 carcinomas, cell cycle stage analysis yielded higher percentages of cells in S and G2M phase for ductal (13% and 12%, N = 22) than for lobular (8% and 7%, N = 8) node-negative carcinomas (p less than 0.002). In ductal carcinomas, lymph node involvement was reflected by higher % G2M values (15%, N = 26) compared with negative cases (12%, N = 22) (p less than 0.05). Ductal node-positive T3-T4 carcinomas (N = 10) revealed a higher % S value (16%) than their T1-T2 counterparts. A correlation between MR and % G2M was established only up to a tumor size of 4.2 cm (r = 0.39, p less than 0.05). A highly sensitive ('H') and a poorly sensitive ('P') subgroup of carcinomas with respect to heparin-induced changes in fluorescence intensity of the G1/0 peak of the DNA aneuploid cell line were identified, as previously shown. These subgroups were here updated with a larger number of carcinomas and were limited to T1-T2 cancers (N = 57). Group 'H' included more younger patients (p less than 0.005), less cases with nodal involvement in ductal carcinomas (p less than 0.05), and lower % G2M values in lobular node-negative cases (p less than 0.05), than group 'P'. DNA diploid cells always existing in DNA aneuploid carcinomas are more sensitive than their aneuploid counterparts (p less than 0.01); however, they strengthen the stratification to 'H' and 'P'. We suggest 'H' carcinomas to be less aggressive than 'P' carcinomas. Small breast carcinomas are recommended to cell kinetic investigations for individualizing adjuvant therapy.

Morphologic and cell kinetic investigations of the cryolesion

An understanding of cryotherapy is based upon knowledge not only about cryobiological, but also about cryomorphologic changes in unaltered and tumorous tissue. 1–6 The morphology of a cryosurgical intervention, which can be registered as a snapshot with the usual morphological techniques, depends on multiple tissue and apparative factors. Thus, general and specific cellular and tissue alterations after local short-term freezing can be detected, e.g., changes in microcirculation, but also cellular and tissue reactions as a defense of the organism, i.e., inflammatory reactions. 4,5,7 Furthermore, ultrastructural changes can be observed using the electron microscope. 8 In comparison with other stimuli like heat, intoxications, or mechanical influences, the time-dependent course of reactions of the organism consequent to such a short-term local freezing is of great interest. The application of cellular kinetic analysis using thymidine autoradiography may help in demonstrating the dynamics of regenerative/reparative events in the organism after freezing. Using morphology as a snapshot and cellular kinetics as a dynamic method, alterations to the immune system of the organism can also be registered that play a decisive role as a response of the organism to noxiousnesses. 9,10 In the following, the morphologic and cellular kinetic findings after single and repeated cryolesions in various tissues are presented, and the consequences of cryolesions vs. thermolesions are discussed.

Cell kinetic investigations in brain tumors studied by serial stereotactic biopsy.

Between July 1983 and July 1986, 109 consecutive patients affected by brain tumors and selected for stereotactic biopsy underwent in vitro investigation of cell kinetics. The potential proliferative activity of these different neoplasms, evaluated as in vitro 3H-thymidine labeling index (LI), has been determined in 46 mature astrocytomas, 19 anaplasic astrocytomas, 25 glioblastomas, 5 cystic craniopharyngiomas, 6 metastatic tumors, 4 primary C.N.S. lymphomas, 3 pineal germinomas and one choroid plexus papilloma. The relevance of LI on prognosis has been evaluated in 35 patients affected by glial tumors with adequate follow-up longer than 2 years. This retrospective study allowed us to demonstrate the prognostic value of LI in mature astrocytoma and anaplasic astrocytoma series. The feasibility of LI determination by in vitro procedure has been demonstrated also in non-neuroectodermal tumors. In craniopharyngioma the LI investigations allowed to demonstrate a peculiar topographic arrangement of cells in S phase.

Typical hyperplasia of the prostate: morphologic, cell kinetic, and immunohistochemical aspects

In histologic, cell kinetic, and immunohistochemical investigations, new aspects in the pathohistogenesis, classification, and differential diagnosis of typical prostatic hyperplasia were demonstrated. In particular, the significance of the immunohistochemical expression pattern of prostatic tissue antigens (PSA, PAP), cytokeratins, and hormonal receptors (ER, PR) as well as proliferation activity with DNA precursors in the pathogenesis of benign prostatic hyperplasia (BPH) and in differential diagnosis of prostatic cancer are discussed. The classification may differentiate histologically and cytologically into typical and atypical, primary and secondary hyperplastic, and atrophic lesions. The term hyperplasia of the prostate should be used instead of “adenoma”.

A Stem Cell Model of Human Tumor Growth: Implications for Tumor Cell Clonogenic Assays

A simple stem cell model of human tumor growth is presented. Three tumor cell populations are predicted: stem, transitional, and end cells. The properties of these cells are discussed in terms of their behavior in currently available technologies for investigation of cell kinetics and for their influence on clinical outcome. Stem cell renewal, transitional cell proliferation, and cell loss are analyzed mathematically to define their influence on the relative proportions of cell populations; it is demonstrated that stem cell renewal has a central role in determining the growth properties of tumors. The impact of a stem cell model on the use of tumor clonogenic assays as predictors of clinical outcome is discussed; opinions are expressed as to the definition of reasonable expectations for current experimental procedures.

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A simple stem cell model of human tumor growth is presented. Three tumor cell populations are predicted: stem, transitional, and end cells. The properties of these cells are discussed in terms of their behavior in currently available technologies for investigation of cell kinetics and for their influence on clinical outcome. Stem cell renewal, transitional cell proliferation, and cell loss are analyzed mathematically to define their influence on the relative proportions of cell populations; it is demonstrated that stem cell renewal has a central role in determining the growth properties of tumors. The impact of a stem cell model on the use of tumor clonogenic assays as predictors of clinical outcome is discussed; opinions are expressed as to the definition of reasonable expectations for current experimental procedures.

An Autoradiographic Study of Cell Kinetics in Epidermis of the Toad Bufo bufo bufo (L)

Under normal conditions, the toad Bufo bufo bufo molts about once a week. It was considered of interest to study whether the rhythmic pattern of cell loss through shedding of a stratum corneum is correlated with a rhythmic production of cells in stratum germinativum. An autoradiographic investigation of cell kinetics in toad skin using [3H]-thymidine and [14C]-thymidine as precursors revealed that cell production in stratum germinativum is a continuous process, and the cell production rate is much higher than is required for replacement of the cells lost with each molt. The parameters measured, i.e., % labeled cells, length of cell cycle, and S-phase, as well as migration of labeled cells from stratum germinativum toward stratum corneum support the assumption of an inhomogeneous cell population in stratum germinativum. A model proposed by Potten form the cellular organization of mouse epithelium provides a useful basis for the interpretation of the results obtained in the present study.

Must initiators come first? Tumorigenic and carcinogenic effects on skin of 3-methylcholanthrene and TPA in various sequences.

Groups of hairless mice were treated with 4 skin applications of 470 nmol 3-methylcholanthrene (MCA) in benzene and 4 of 20 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) in various sequences, twice a week, together and separately. Three days after the last application, cell kinetic investigations were made comprising the counting of basal and suprabasal cells, the assessment of hyperplasia, the mitotic rate by the stathmokinetic method, the labelling index and the specific activity of DNA after injection of a dose of [3H]dT, and the determination of percentage of cells in each cell-cycle phase by flow cytometry. These studies showed that various treatment schedules with 4 applications stimulated proliferation and caused epidermal hyperplasia, but there was no significant difference between the groups in degree of growth stimulation. There was a significantly higher tumour production by all the combinations than by MCA alone. It was of no significant importance for the tumour production whether the 4 applications of MCA came before or after the 4 of TPA. Alternating treatment (MCA-TPA, etc.) seemed to give a higher tumour risk than the other treatment sequences. The consequences of these results for the two-stage theory of carcinogenesis (stating that initiation must come first) are discussed, and it is concluded that (at least under the experimental conditions used here) initiation does not need to come first for a good tumour yield.

Morphological and cell kinetic investigations of wound healing of rat kidney after local freezing

The effects of cryonecrosis in rat kidney after single and repeated local freezing were studied by morphological and cell kinetic methods. Shortly after single and repeated cryolesions a well-demarcated coagulation necrosis developed which was replaced by granulation tissue. 4 weeks after the second freezing only a small fibrous scar was visible. Cell analysis revealed no significant differences between single and repeated cryolesions. Autoradiographically, however, it was shown that the labelling index of fibroblasts in the granulation tissue was higher after repeated than after single cryolesions. The proliferation maxima of epithelial cells were delayed for 24 h. After 4 weeks the cell proliferation again had reached normal values. Hardly any differences between wound healing after single and after repeated cryolesion were found. Thus, repeated is advisable and no complications should be expected.

A theoretical consideration of some biological parameters involved in cell kinetic investigations of oral leukoplakia and abnormal states in stratified squamous epithelium.

The correct interpretation of cell kinetic data for normal epithelia requires a consideration of several interrelating factors. In abnormal states, such as leukoplakia, the lack of appropriate data for these parameters makes such interpretation difficult. Consequently, an altered mitotic index need not indicate an abnormality of cell proliferation.

Untersuchungen zum Proliferationsmuster von drei Plattenepithelkarzinomen mit einer modifizierten autoradiographischen in vitro-Methode

Common in vitro methods for autoradiographic studies lead to a destruction of the histological structures of the tumor specimen which were for instance taken from biopsy in man. We report about a modified autoradiographic in vitro method for incubating human tumor material in H-3-thymidine in a special oxygen chamber. This method allows to use intact tumor slices and maintaines the histological appearance for the cell kinetic investigations. The proliferation rate of three different squamous cell carcinomas of the oropharynx were studied and one tumor was xenografted on thymusaplastic nude mice.

Histologische und proliferationskinetische Untersuchungen zum Wachstum der Rattenprostata in verschiedenen Lebensaltern

Histologic and cell-kinetic investigations were performed on the developing prostate of growing rats. The ages ranged from 1 day to 15 months. The most intensive cell proliferation is observed within the first 2 weeks after birth. Thereafter, the cell proliferation decreases continually as the cellular differentiation of the prostatic glandular epithelium increases to form well-differentiated secretory cells. The second peak of cell proliferation occurs with the beginning of sexual maturation and an elevated testosterone production. But the rapid increase of prostatic weight at that time is mainly caused by an increased formation of secretory and interstitial tissue fluids. The different lobes of the adult prostate all exhibit the same cellular proliferation pattern. The prostate belongs to the organs with stable growth pattern and an only small cellular turnover. Mitotic figures are extremely rare under normal conditions. But this low cellular proliferative activity may be easily changed, in contrast to other organs with stable tissue and steady-state growth, i.e., liver and kindney, in dependence of hormone levels.

Cell-kinetic investigations on the epidermis of uninvolved skin of patients suffering from seborrheic eczema.

Cell-kinetic investigations on the epidermis of uninvolved skin of patients suffering from seborrheic eczema.