PCNA bearing structures are retained in apoptotic phase of childhood ALL cell cycle.

BACKGROUND: Drug resistance to DNA directed therapy may depend on proliferative as well as apoptotic cell fraction. PCNA/Ki67 ratio excess, possibly reflecting DNA excision repair, is of additional interest to drug resistance in MTT testing. The cell cycle phase/antigen expression pattern in childhood acute lymphoblastic leukemia (ALL) is not known. AIMS: To study the relationship between nuclear expression of PCNA, Ki-67 and Frag-EL positivity in childhood ALL. METHODS: 1.3.1. Study Groups. Diagnostic bone marrow trephine biopsies of 32 consecutive unselected cases of childhood ALL were included in the study. 1.3.2. Immunohistochemistry. Commercially available Moab PCNA (PC10, DAKO, USA), Ki-67 (MM1, NovaCastra, UK) were used to label cycling cells in routinely processed 5 microns paraffin sections. 1.3.3. In-Situ Labelling of Apoptotic Cells. The 3'-OH ends of apoptosis specific DNA fragments were labelled in-situ on subsequent 10 microns sections (Frag-EL, CalBiochem, USA). 1.3.4. Quantitation. After blinding and randomisation, 10 systematic random fields of > 20 nuclei and nuclear size bias correction was used to determine positive nuclei fraction. RESULTS: While the sum of apoptotic and proliferative cell fraction (Ki-67 + Frag-EL%) equalled 100% in 5/32 cases, PCNA expression into at least the early phases of apoptosis ([%PCNA-%K-67] > [100-%Frag-EL] was found in 17/32 cases. CONCLUSIONS: PCNA/Ki67 ratio excess may not reflect DNA excision repair activity but rather slow degradation of antigen bearing structures limiting relevance to drug resistance study.

Apoptosis corrected proliferation fraction in childhood ALL is related to karyotype.

BACKGROUND: Tumour doubling time, a parameter in drug sensitivity testing, reflects both cell proliferation and apoptosis. Variable apoptosis fractions may explain the poor correlation of S-fraction and drug response. DNA aneuploidy (reflecting intrinsic DNA instability) may, by increasing apoptosis, affect drug response. AIM: To assess the relationship between apoptosis corrected proliferation fraction and DNA ploidy in childhood acute lymphoblastic leukemia (ALL). METHODS: 1.3.1. Study Groups. Thirty two consecutive, unselected diagnostic cases of childhood ALL were included in the study. 1.3.2. Karyotype. A normal karyotype was found in 15 cases (7M, 8F, age 8 m-12 yrs); high hyperdiploid aneuploidy (DNA index > 1.5) was found in 7 patients (1M, 7F, age 3-12 yrs) whereas complex karyotypic anomalies, but with 2n or near 2n DNA were present in 10 patients (7M, 3F, age 1 y 7 m -16 yrs). 1.3.3. Proliferation Fraction Assessment. Immunocytochemical demonstration of S-phase associated nuclear expression of the Ki-67 antigen (MM1, NovaCastra, UK). 1.3.4. Apoptosis Fraction Assessment. Binding of a horse radish peroxidase labelled DNA probe for the 3'-OH ends of apoptosis derived Klenow fragments (Frag-EL, CalBiochem, USA). 1.3.5. Quantitation. Computer assisted image analysis (Quantimet 570C), of 10 systematically random fields of a minimum of 20 nuclei each. A nuclear size bias correcting counting frame and rule were used to correct for cell proliferation associated nuclear volume increase and for the expected nuclear volume reduction resulting from apoptosis. RESULTS: Corrected for apoptosis, proliferation fraction was highest (mean 57.5%, range 1-100) in poor prognosis, complex karyotype anomalies. Good prognosis, high hyper diploidy showed significantly lower proliferation rates (mean 24.7%, range 12-40) (p < 0.01, t-test). CONCLUSION: Apoptosis corrected cell proliferation rate in childhood ALL is not independent of karyotype abnormality which may partly explain a relation to therapy response and prognosis.

Differential kinetics of drug resistance in human leukaemic cells measured by SCGE/CLSM.

BACKGROUND: New analogues of DNA directed chemotherapy moieties are available for comparative efficacy testing in human neoplastic disease. In addition to MTT testing direct assessment of DNA excision repair activity after direct exposure of marrow cells may provide information on relative DNA effects in vitro. AIMS: To assess the ability of SCGE/high resolution CLSM to detect differences in drug resistance between human neoplastic cell lines in the DNA excision repair response to chemotherapy. METHODS: Eight human leukaemia samples (4 childhood, 4 adult) were exposed to 1 hour of single concentrations of daunorubicin, DaunoXome (courtesy NeXstar Pharmaceuticals Inc, USA), cyclophosphamide and 4-hydroperoxycyclophosphamide (4-HC, courtesy Dr. M. Colvin, Duke University, USA), followed by SCGE/high resolution CLSM with quantitation of total excised DNA. Differences between cases/drug moieties/exposures were analysed. RESULTS: Although generally equal effect dose levels for DaunoXome were lower than for standard daunorubicin, patients/individual neoplastic cells differed considerably in optimal dose levels. Conventional cyclophosphamide in comparison to 4-HC showed inconsistent results indicating considerable differences in the level of drug resistance to the conventional product. CONCLUSIONS: Direct testing for drug resistance patterns in DNA directed drug moieties by SCGE/CLSM reveals individual variability of human malignant cell lines warranting comparison with results of MTT testing and in-vivo patient response.

Demonstration of differences in drug resistance by direct testing of DNA excision repair activity following standard and liposomal daunorubicin exposure in normal paediatric marrow using high resolution CLSM.

BACKGROUND: High resolution Confocal Laser Scanning Microscopy (CLSM) may be applied to testing of drug resistance in vitro in clinical setting. Rapid analysis of DNA damage by precise quantitation of excised DNA in bone marrow samples exposed to potential treatment moieties directly after isolation but the relative sensitivity of the integrated method is as yet untested. AIMS: To test the clinical applicability of SCGE/high resolution CLSM for differences in drug resistance in marrow cells. METHODS: Cells from normal bone marrow samples were exposed for identical periods and at 4 concentrations to either 1 hour of standard Daunorubicin (.5, 1, 1.5, 2 micrograms/ml) or 8 hours DaunoXome (courtesy of NeXstar Inc, USA) (.05, .1, .15, .2 microgram/ml). After 2 and 6 hours recovery, cells were harvested for SCGE, randomization, analysis of tail length, total excised DNA and fragment size distribution using high resolution CLSM. RESULTS: Tail length and fragment size distribution was not, but total excised DNA was significantly increased after 0.1 microgram/ml Liposomal Daunorubicin (DaunoXome) compared to 1.0 microgram/ml Daunorubicin. CONCLUSION: SCGE/high resolution CLSM effectively demonstrated differences in Daunorubicin resistance of human marrow cells to alternative formulations. The method has potential for use in clinical testing of neoplastic cell drug resistance.

High resolution confocal laser scanning microscopy analysis of DNA excision repair capability in small volume marrow samples exposed to DNA directed treatment moieties.

BACKGROUND: Assessment of resistance to drug moieties in tissue culture is complicated by limited sample, clonal selection and alteration of cycling fraction and cycle duration in clonally mixed lesions. DNA damage assessment by single cell gel electrophoresis (SCGE) of excised DNA is limited by non-linear analysis in fluorescent light microscopy. Confocal Laser Scanning Microscopy (CLSM) with high N.A. magnification allows for quantitation of total excised DNA fragment size distribution but is still limited by the large volume required for labour intensive SCGE, precluding multi-exposure clinical testing. AIMS: To optimise sample requirement for SCGE and CLS. METHODS: Standard slide mounted bed gels were punched with multiple coded 6 mm wells and filled with suspensions of cells subjected to drug/concentration variations. After SCGE, 30 microns frozen sections were prepared of each well and mounted in ethidium bromide solution on multi-well hydrophilic slides to allow for short working distance of high resolution CLSM in a Zeiss Axiovert L410 SM. Testing for feasibility, reproducibility and consistency used both cultured standard leukaemic cell lines, normal human control marrow and clinical samples. RESULTS AND CONCLUSION: Multiple well SCGE followed by frozen section, high resolution CLSM allows for rapid analysis of high numbers of multiple drug exposure permutations clinically required.

Aortic valve grafts in the rat: evidence for rejection.

OBJECTIVE: The histopathologic changes of fresh rat aortic valve allografts over time and the effect of cryopreservation were examined. METHODS: Fifty-six syngeneic and allogeneic rat aortic valves were transplanted, either fresh or after cryopreservation, and then at different time points they were explanted and histologically examined in a blinded fashion. RESULTS: Histopathologic changes in the first week are similar in syngeneic and allogeneic grafts. Fresh syngeneic grafts and leaflets retained normal structure up to 56 days. Allogeneic grafts showed retrovalvular thrombus formation with leaflet ghosts and neointimal proliferation. Cryopreservation did not alter this process. CONCLUSIONS: Cardiac allograft valves in the rat model undergo changes that are characteristic of cell-mediated rejection and lead to valve failure.