Biomedical subjects

D P Calkins

Publications and source records attributed to D P Calkins.

7 recordsLinked to original sources

PubMed1997

Proliferation and hypoxia in human squamous cell carcinoma of the cervix: first report of combined immunohistochemical assays.

PURPOSE: To characterize the distribution of hypoxia and proliferation in human squamous cell carcinoma of the cervix via an immunohistochemical approach prior to initiation of therapy. METHODS AND MATERIALS: Patients with primary squamous cell carcinoma of the cervix uteri received a single infusion of the 2-nitroimidazole, pimonidazole (0.5 g/m2 i.v.), and 24 h later punch biopsies of the primary tumor were taken. Tissue was formalin fixed, paraffin embedded, and sectioned for immunohistochemistry. Hypoxia was detected by monoclonal antibody binding to adducts of reductively activated pimonidazole in malignant cells. Staining for endogenous MIB-1 and PCNA was detected in tumor cells via commercially available monoclonal antibodies. Point counting was used to quantitate the fraction of tumor cells immunostained for MIB-1, PCNA, and hypoxia marker binding. RESULTS: Immunostaining for pimonidazole binding was distant from blood vessels. There was no staining in necrotic regions, and only minimal nonspecific staining, mostly in keratin. In general, cells immunostaining for MIB-1 and PCNA did not immunostain for pimonidazole binding. Cells immunostaining for MIB-1 and PCNA showed no obvious geographic predilection such as proximity to vasculature. Quantitative comparison showed an inverse relationship between hypoxia marker binding and proliferation. CONCLUSIONS: Immunohistochemical staining for pimonidazole binding is consistent with the presence of hypoxic cells in human tumors and may be useful for estimating tumor hypoxia prior to radiation therapy. Immunostaining for pimonidazole binding is an ideal complement to immunohistochemical assays for endogenous proliferation markers allowing for comparisons of tumor hypoxia with other physiological parameters. These parameters might be used to select patients for radiation protocols specifically designed to offset the negative impact of hypoxia and/or proliferation on therapy. The inverse relationship between pimonidazole binding and proliferation markers is a preliminary result requiring verification.

Adult↗

PubMed1995

Distribution of hypoxia and proliferation associated markers in spontaneous canine tumors.

The therapeutic response of malignant tumors depends on a number of factors associated with tumor microenvironments including the possibility that these microenvironments change during treatment. Two factors, tumor hypoxia and cell proliferation, have been examined in spontaneous canine tumors undergoing multifraction radiation therapy. The approach utilizes immunohistochemical analyses of hypoxia (CCI-103F) and proliferation associated (PCNA) antigens in biopsy samples taken before and after 5 daily fractions of 3 Gy (total dose 15 Gy). The tissue samples were formalin-fixed and paraffin-embedded for the immunohistochemical study. Immunostaining of the sections for PCNA and hypoxia marker reveals little or no overlap when the analysis is made prior to irradiation. An increased degree of overlap seems to occur after 15 Gy but the situation is complicated by a change towards more diffuse PCNA immunostaining in the cells of the irradiated tissues.

Animals↗

PubMed1993

The relationship between proliferative and oxygenation status in spontaneous canine tumors.

PURPOSE: Immunocytochemical markers have been applied to biopsy specimens from spontaneous canine tumors to assess the prevalence and spatial distribution of proliferating and hypoxic cells, and their "geographic" relationship to each other. Both types of cells have been implicated in the failure to locally control human tumors treated with radiation and chemotherapy. METHODS AND MATERIALS: For the detection of hypoxic cells, a rabbit polyclonal antibody raised against a protein-bound, hexafluorinated, 2-nitroimidazole, designated CCI-103F, was used. The unmetabolized drug must first be injected into the dog to allow time for hypoxic metabolism and cellular binding to occur. For the detection of proliferating cells, a mouse monoclonal antibody raised against an endogenous nuclear protein, the "proliferating cell nuclear antigen," or PCNA, was used. This protein is expressed in most actively proliferating cells, but not in quiescent ones. An indirect immunostaining technique was used to visualize these markers in the tissue sections, and image analysis was used to estimate the area fraction of positive staining in representative, low magnification microscope fields. RESULTS: Tumors with both high and low hypoxic and proliferative area fractions have been identified. No systematic relationship between the prevalence of the two markers, nor of the relationship between tumor grade and proliferative fraction, could be established. Staining with the proliferation marker was more commonly found near blood vessels, but some "nests" of tumor cells apparently distant from vasculature contained many proliferating cells. Staining with the hypoxia marker tended to be distant from the vasculature and/or bordering regions of tumor necrosis, but some labeled cells appeared near blood vessels, and in the absence of necrosis. Staining of sequential sections, one with the proliferation marker and one with the hypoxia marker, indicated that the two cell populations overlapped to varying extents. Some incidental staining of canine normal tissues with both the proliferative and hypoxia markers was observed as well. CONCLUSION: The immunochemical marker approach promises to be a useful tool to increase both our basic understanding of tumor physiology and the complex nature of tumor heterogeneity.

Animals↗

PubMed1991

Evaluation of flow cytometric methods for determining population potential doubling times using cultured cells.

Various methods have been proposed for determining the potential doubling times (Tpot) of mammalian cell populations by using flow cytometric techniques after labeling the cells with bromodeoxyuridine (BrdUrd). We show here that, in a well-defined in vitro system where multiple time measurements are possible, all the methods give similar results that are close to the true population doubling time. Of ultimate interest, however, is the accuracy of determination of Tpot from a single time point. In this paper we compare the accuracy and precision of the methods in making such determinations at different times after labeling. The relative movement (RM) of BrdUrd-labeled cells that have not divided at the time of assay allows for computation of the length of S phase (Ts). The precision of estimation of Ts was enhanced when a quantity, v (a function of the fraction of BrdUrd-labeled divided and the fraction of BrdUrd-labeled undivided cells), was used to estimate the initial intercept of RM. Furthermore, calculation of Tpot from the formula, Tpot = ln(2) Ts/v, gave values closest to the observed population doubling time. It is suggested that the use of RM with v be the analytical method of choice for the calculation of Tpot from single time-point observations, preferably made at times between the length of the G2 and M phases (TG2M) and Ts.

Animals↗

PubMed1990

Improved method for computing potential doubling time from flow cytometric data.

Relative movement methods use the timed progression of the mean fluorescence of cells which have been labeled with monoclonal antibodies against bromodeoxyuridine and displayed with bivariate flow cytometry according to DNA and label content to compute duration of DNA synthesis, TS. The relative movement is the difference of the mean DNA fluorescence of the labeled undivided cells from the G1 channel relative to the difference between the G1 and G2M channels. In this communication, we show how to extend this method to compute the potential doubling time, Tpot, the time required for a population of cells to double, given quiescent cells but no cell loss. A quantity v is introduced that is a function of the fraction of labeled divided cells and the fraction of labeled undivided cells. We show that v is independent of time and is equal to ln(2)Ts/Tpot so that Tpot (equal to ln(2)Ts/v) can be directly found from the information available in computing the relative movement. The method is applied to Chinese hamster ovary cells to demonstrate its utility.

Animals↗

PubMed1990

Effects of beta-all-trans retinoic acid on growth, proliferation, and cell death in a multicellular tumor spheroid model for squamous carcinomas.

The growth of multicellular tumor spheroids, MTSs, from squamous carcinoma line MDA 886Ln was inhibited by beta-all-trans retinoic acid (RA). Inhibition occurred within 3 to 5 days of treatment, and MTS size then remained static for up to 2 weeks. Although their growth stopped, 10-day-treated MTSs incorporated [3H]thymidine into trichloroacetic acid-precipitable material, and the [3H]thymidine labeling index, determined by autoradiography, was equivalent between control and RA-treated MTSs. Bivariate flow cytometric analysis of bromodeoxyuridine-labeled MTSs showed equivalent S phase progression of labeled cells over an 8-hour chase. MTS growth stasis was not related to RA-induced cell cycle effects. Monitoring of MTSs for cell sloughing showed no significant cell shedding that could account for stasis. Quantitation of cell number and DNA content per MTS showed an RA-induced decrease. This was confirmed by histological analysis, which demonstrated the temporal appearance of acellular areas. MTS growth statis is thus related to an RA-induced cell loss in this MTS model for squamous carcinomas.

Autoradiography↗

PubMed1990

The accuracy of fine needle aspiration biopsy for flow cytometric determination of tumor DNA content.

The use of fine needle aspiration (FNA) to obtain a diagnosis of malignancy is established in the practice of oncology, but there is little information on its accuracy in sampling tumor DNA content. We therefore compared flow cytometric DNA data obtained from FNA-derived samples with that obtained after digestion of the same murine tumor from which the aspirates had been taken. Fifteen female C3Hf/Kam mice were implanted with MCA-29 tumor cells from the same source tumor. MCA-29 is a multiploid mammary adenocarcinoma with two aneuploid populations (DNA Index of A = 1.67, B = 1.89). The tumors were grown to a mean size of 8.6 mm. After sacrifice, three FNAs were performed on each tumor, following which the whole tumor (WT) was excised and homogenized. All FNA and WT samples were digested with 0.04% pepsin and the nuclei stained with propidium iodide in preparation for flow cytometry. DNA histograms of the aspirates were compared with the corresponding WT histograms. Any single FNA detected population A in all (100%) cases and detected the less prominent population B in 94.3% of instances. Any single FNA was able to detect the same populations that were present in the whole tumor in 95.4% of cases, while the set of three aspirates matched the corresponding WT in 100% of cases. We conclude that FNA DNA histograms are accurate for the assessment of ploidy, but that in order to ensure detection of all tumor populations present, multiple aspirates are needed.

Animals↗