Assessment of proliferating cell nuclear antigen (PCNA) in breast cancer using anti-PCNA PC10 and 19A2: correlation with 5-bromo-2'-deoxyuridine or tritiated thymidine labeling and flow cytometric analysis.

We compared immunohistochemical staining by two monoclonal antibodies, PC10 and 19A2, to standard methods for cell proliferation, 3H-TdR or BrdU labeling index (S-LI) and S-phase fraction by flow cytometry (S-flow). One hundred thirty-two breast carcinomas were studied retrospectively using formalin fixed, paraffin embedded archival tissues on which S-LI and S-flow had been obtained originally. Percentages of tumor cells positive with PC10 and 19A2 correlated well (r = 0.736, p < 0.0001), although the mean marking index for 19A2 was lower and closer to reference measurements than the mean PC10 index. Correlations between PC10 or 19A2 vs. S-LI, S-flow or DNA ploidy (DNAI) were significant in a group of 64 tumors obtained between 1988 and June 1992, and poor in another group of 68 tumors obtained between 1985 and 1988, suggesting deterioration of stainability with prolonged storage. Discrimination of faint staining from negative nuclei was difficult on PCNA stained sections. Carnoy fixation did not improve results over those fixed with formalin. S-flow and S-LI predicted relapse-free survival, but PCNA indices did not. We conclude that PC10 and 19A2 immunostaining of formalin or Carnoy fixed archival breast cancer tissue correlated with reference measures of proliferation only in cases of short storage periods. Although statistically significant, levels of correlation were too low to use PCNA indices for prognosis.

Bromodeoxyuridine labeling for S-phase measurement in breast carcinoma.

BACKGROUND: The S-phase fraction relates to proliferation, an important determinant of tumor behavior, and has been measured most accurately with the DNA precursor tritiated thymidine (TT). The TT labeling index (LI) is a strong stage-independent prognostic indicator for breast carcinoma. The thymidine analogue 5-bromodeoxyuridine (BrdU) is also incorporated into DNA and has the advantage over TT of immunohistochemical detectability rather than requiring autoradiography, but it is less well studied in breast carcinoma. This report demonstrates the equivalence of TT and BrdU LI and explores the relationships between LI and other biologic measurements. METHODS: The LI of 234 consecutive breast carcinomas were measured with TT as was a subsequent series of 450 cases with BrdU, both by incubation in vitro. RESULTS: The mean BrdU LI was 6.4 +/- 0.3% in comparison with 6.9 +/- 0.4% in the prior TT series. LI was unaffected by storage for 24 hours at 4 degrees C before labeling with BrdU. The BrdU and TT LI both correlated: (1) positively with tumor size, histologic type, nuclear size, the number of axillary metastases, the level of DNA ploidy, and the percent S-phase by flow cytometry and (2) negatively with the age of the patient and the levels of estrogen receptor and progesterone receptor measured either by ligand binding or by immunohistochemistry. CONCLUSIONS: BrdU labeling in vitro was an advantageous method for measuring S-phase fraction in breast carcinoma that produced results comparable to those from TT labeling. It should be equally effective for breast cancer kinetic classification and prognosis and is a suitable standard to evaluate newer methods for measuring cellular proliferation.

Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling.

We labeled active S-phase cells in primary breast carcinomas with a modified 5-bromo-2'-deoxyuridine (BrdU) procedure using a silver-enhanced colloidal gold visualization step. Separate samples of 29 tumors were labeled with BrdU or tritiated thymidine ([3H]-dThd), and the labeling indices (LI) from the two methods were equivalent (Spearman's correlation coefficient = 0.96). Three breast carcinomas were incubated in various mixes of both BrdU and [3H]-dThd and developed sequentially for each. Paired photomicrographs showed that the same nuclei were labeled by either precursor. The in vitro method yielded LIs similar to those reported after in vivo pulse BrdU labeling for tumors of the central nervous system. The BrdU LI correlated significantly (r = 0.76, p less than 0.001) with % S-phase by DNA flow cytometry in 33 breast carcinomas. The BrdU labeling method is simpler and more rapid than the [3H]-dThd procedure (1-2 days for completion for the former, 7-10 days for the latter), and it provides an equivalent measurement of proliferative index.

In situ patch-clamp recording of calcium-activated potassium channels from an identified leech neuron.

The paired anterior lateral giant cells of the leech Haementeria have only two active voltage gated ionic currents. We took advantage of this simple complement of ionic currents to investigate the single channel properties of this cells' calcium-activated K+ current (I(K,Ca) in situ. Cell-attached patch recordings showed large, bursting events with a conductance of approximately 90-100 pS which had extrapolated reversal potential consistent with K+ events. The channel open time distribution was well described by a single exponential process while the channel closed times were bi-exponentially distributed. The results show that the single channel properties of I(K,Ca) in annelids closely resemble those of similar currents described in vertebrates.

Binding of calmodulin to the microfilament network correlates with induction of a macrophage tumoricidal response.

Induction of mouse peritoneal macrophage cytotoxicity against SV3T3, a line of virally transformed mouse cells correlated with the distribution of cytoplasmic calmodulin in the macrophages. The organization of the cytoskeleton was examined by fluorescent microscopy and by transmission electron microscopy, using immunogold tagging after Triton-X-100 (TX-100) extraction of the macrophages. Macrophages that had been activated to a tumoricidal state in vivo by vaccinia virus or in vitro by lymphokine stimulation displayed cytoskeletal networks that were more extended and weblike than did resident macrophages. The organization of microfilaments and microtubules in the cytoskeleton was displayed by using either anti-actin or anti-tubulin. Immunogold labeling of tumoricidal macrophage cytoskeletons with anti-calmodulin revealed strong binding to the microfilament network and no binding to microtubules. Anti-calmodulin reacted weakly with the cytoskeletal network of resident macrophages, and this was not demonstrably greater than the reaction with normal sheep serum. However, resident macrophages displayed a high density of calmodulin (CAM) associated with unidentifiable structures in the perinuclear region when reacted with anti-calmodulin. These characteristic distributions of CAM in resident and activated macrophages was confirmed by immunofluorescence. The total and cytoskeletal-associated amounts of calmodulin per unit of protein were determined by radioimmune assay and 125I labeling followed by SDS-PAGE. No statistically significant differences were detected between resident and activated macrophages in either the total cell or cytoskeleton fractions. In summary, our results suggest that induction of tumoricidal activity of mouse peritoneal macrophages correlates with the translocation of calmodulin to the microfilament network of the cytoskeleton.

The effect of preparative protocol on the cytoskeleton ultrastructure observed in extracted whole-mount BHK cells.

Extracted BHK cells (baby hamster kidney) were prepared for electron microscopy by air-drying (with Freon 113), critical-point-drying, and freeze-drying. Variations in the drying procedures had a marked effect on the resultant cytoskeleton ultrastructure. Air drying had to be done in a Freon-saturated atmosphere, residual water had to be removed from the dehydrating solutions and carbon dioxide for critical-point-dried specimens, and freeze-drying had to be done at temperatures lower than -90 degrees C. Failure to exercise these precautions resulted in a cytoskeleton ultrastructure artifact, possibly caused by fusion of the cytoskeleton filaments.