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 vitro immunohistochemical localization of S-phase cells by a monoclonal antibody to bromodeoxyuridine

Thin viable slices of normal or pathological human tissues were incubated in vitro with bromodeoxyuridine (BrdU). Later, cryostatic sections and histological sections from the same samples embedded in paraffin were examined by an immunohistochemical method using a monoclonal antibody anti-bromodeoxyuridine (anti-BrdU-MAb): on both cryostatic and histological sections, the nuclei of the S-phase cells proved positive. The optimization of the technique depends on the concentration of bromodeoxyuridine in the culture medium (160 microM), the duration of incubation (not less than two h), the method of DNA denaturation (2N or 4N HCl) and the dilution of the anti-BrdU-MAb (1:50). In vitro, immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections.     

In vitro labeling of solid tissues with tritiated thymidine for autoradiographic detection of S-phase nuclei.

In vitro measurement of the thymidine labeling index (TLI) of solid tissues requires hyperbaric oxygenation and is potentiated by blockade of thymidylate synthetase by 5-fluoro-2'-deoxyuridine (FUdR) to favor uptake of tritiated thymidine (3H-TdR). Hyperbaric oxygenation can be achieved in a simple system through injection of oxygen into rubber-stoppered test tubes. Incubations are carried out in Hanks' balanced salt solution in a shaker bath at 37 C for 2 hours; an FUdR concentration of approximately 1 micron is optimal. Autoradiographic exposure for 1 week or less is sufficient for TLI measurements on human tissues. With 3 to 4 atmospheres oxygen tension, incorporation of 3H-TdR is sufficient for TLI measurement throughout slices of tissue cut 1 mm thick or less. Mincing of tissue is not necessary, and the anatomic continuity seen in ordinary histological preparations is preserved. A gradient of labeling intensity is present from the surface to the interior of the tissue, but sufficient intensity of labeling for detection of DNA synthesis can be achieved in the interior of the section. The gradient can be reduced only slightly by prior incubation in 3H-TdR with hyperbaric oxygen at 0 C. The method permits TLI measurements on the same specimens, including needle biopsies, that are used for pathologic diagnosis.     

Rapid in vitro bromodeoxyuridine labeling method for monitoring of therapy response in solid human tumors

In vitro bromodeoxyuridine (BrdUrd) incubated single-cell suspensions obtained from solid tumors were fixed on slides for subsequent sample processing. As dispersal of nuclei largely was avoided, only small amounts of cells were needed for examination. The sensitivity of detecting even low BrdUrd incorporation rates could be improved by treatment with intense DNA denaturation conditions. This technique was applied to monitor cytokinetic response to chemotherapy and radiation in oral carcinomas by analysing biopsies taken consecutively in the course of treatment. By combining BrdUrd labeling and DNA flow cytometry, cells arrested in S phase easily could be distinguished from cells showing continuous proliferation.     

In vitro labeling of beta-apolipoprotein with 3H or 14C and preliminary application to turnover studies.

3-H- or 14-C-labeled methyl groups were introduced into apolipoproteins of human and pig low density lipoproteins (LDL). 98% of the label was recovered in the apoprotein of radiomethylated LDL. Such methylated lipoprotein was compared with the corresponding unlabeled LDL with respect to its electrophoretic and immunochemical properties, and its behavior in the analytical ultracentrifuge. The data demonstrated that neither the human nor pig LDL underwent gross changes as a result of methylation. The applicability of radiomethylated pig LDL as a tracer for studying the turnover of LDL in pigs was examined. The results showed that the behavior of unscreened and screened 3-H-labeled LDL was similar. The LDL disappeared with an initial t1/2 of 1.1 hr and a later t1/2 of 30 hr. These values agreed well with those reported for radioiodinated LDL. The technique of radiomethylation of lipoprotein may afford an advantage over radioiodination as it may label peptides that do not have tyrosine.     

Double labeling and in vitro versus in vivo incorporation of bromodeoxyuridine in patients with acute nonlymphocytic leukemia

A monoclonal antibody against bromodeoxyuridine (BrdUrd) was produced, and a rapid slide technique (RPMB technique) was developed for the estimation of S-phase cells in a population using this antibody. Bone marrow cells from patients with acute nonlymphocytic leukemia (ANLL) were studied by both the RPMB technique and tritiated thymidine (3HdThd) labeling index studies. The percentage of S-phase cells obtained by each method was compared in 50 samples, and the correlation coefficient was r = 0.89. A "double label" method is also described in which cells were simultaneously incubated with either BrdUrd and 3HdThd or BrdUrd and tritiated cytosine arabinoside (3HAra-C). The samples were first processed by the RPMB technique and then by autoradiography. Results showed only black grains overlying the nuclei of fluorescent cells in each group. An automated microphotometer was used to quantitate grains and fluorescence from each cell. This demonstrated an almost direct relationship between grains and fluorescence from BrdUrd + 3HdThd slides, whereas different patterns of relationship were noted from BrdU + 3HAra-C slides of leukemic patients. Their implications are discussed in the text. Finally, intravenous infusions of BrdUrd was given to five leukemic patients. S-phase cells were recognized distinctly within 5 min of starting the infusion. The percentage of S-phase cells was almost identical from in vivo and in vitro samples. Various possibilities of studying the biological behavior of acute leukemias and analyzing cell cycle characteristics are discussed.     

In vitro triiodothyronine binding to non-histone proteins from rat liver nuclei

$\text{In vitro}$ incubations of non-histone proteins from rat liver nuclei with labelled L-3, 5, 3′ triiodothyronine demonstrate the existence of high affinity, limited capacity binding sites for the hormone in this protein group; the affinity was found identical for triiodothyroacetic acid and lower for L-thyroxine. Binding ability was highly temperature dependent. At 4°C, the rate constant of association was 0.9 × 10⁷ M^?1 h^?1 and the rate constant of dissociation was 0.015 h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 1.6 and 5 × 10^?9 M. The maximum binding capacity was 10^?13 moles of L-3, 5, 3′ triiodothyronine per 100 μg non-histone proteins or 6000 hormone molecules per nucleus. Protein binding had a half-life of 20 hours at 4°C, in the absence of hormone, but was found to be very stable in the presence of hormone.     

In vitro labeling of peroxisomal cholesterol with radioactive precursors

Peroxisomes isolated from rat liver were incubated with [³H]squalene and [³H]mevalonate and the subsequent incorporation of radioactivity into cholesterol studied. The isolated lipids became labeled after incubation with both precursors. In contrast to findings with microsomes, trypsin and detergent treatment of peroxisomes did not influence the rate of cholesterol synthesis. In addition, the luminal content of peroxisomes could alone mediate this synthetic process. Upon treatment of rats with various inducers of peroxisomes and of the endoplasmic reticulum, as well as upon feeding with cholesterol and cholestyramine, large differences in the pattern ofin vitro incorporation of [³H]mevalonate into the cholesterol of peroxisomes and microsomes were observed. Injection of this precursor also resulted in high initial labeling of peroxisomal cholesterolin vivo. These experiments indicate that cholesterol synthesis may also occur in peroxisomes.     

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.     

In vitro labeling of proteins by reductive methylation: Application to proteins involved in supramolecular structures

Actin and tropomyosin, purified from both muscle and brain, and α-actinin, purified from muscle, have been labeled in vitro by reductive methylation to specific activities of greater than 10⁵ dpm/μg protein. Actin so modified bound DNase I and polymerized identically to unmodified actin. Furthermore, the spectral properties of actin did not change after labeling. The interactions of labeled tropomyosin and α-actinin with F-actin were nearly identical to those of the unmodified proteins. These modified proteins comigrated with their unmodified counterparts in both SDS-containing polyacrylamide gels and isoelectric focusing gels. The labeled actin was quantitatively extracted from SDS-containing polyacrylamide gels (yield > 98% of radioactivity applied demonstrating that all of the radioactivity was protein bound. The reductive methylation procedure worked well at pH 8.0–8.5 in either pyrophosphate buffer or Bicine buffer using formaldehyde with [³H]-sodium borohydride as the reducing agent. The procedure could also be performed at pH 7.0 in phosphate buffer using [¹⁴C]-formaldehyde with sodium cyanoborohydride as the reducing agent. Proteins so labeled are ideal for use in quantitative experiments involving protein-protein interactions.     

In vitro labeling of hydroxyapatite minerals by an engineered protein

Biological and biomimetic synthesis of inorganics have been a major focus in hard tissue engineering as well as in green processing of advanced materials. Among the minerals formed by organisms, calcium phosphate mineralization is studied extensively to understand the formation of mineral-rich tissues. Herein, we report an engineered fusion protein that not only targets calcium phosphate minerals but also allows monitoring of biomineralization. To produce the bi-functional fusion protein, nucleotide sequence encoding combinatorially selected hydroxyapatite-binding peptides (HABP) was genetically linked to the 3' end of the open reading frame of green fluorescence protein (GFPuv) and successfully expressed in Escherichia coli. The fluorescence and binding activities of the bi-functional proteins were characterized by, respectively, using fluorescence microscopy and quartz crystal microbalance spectroscopy. The utility of GFPuv-HABP fusion protein was assessed for both time-wise monitoring of mineralization and the visualization of the mineralized tissues. We used an alkaline phosphatase-based reaction to control phosphate release, thereby mimicking biological processes, to monitor calcium phosphate mineralization. The increase in mineral amount was observed using the fusion protein at different time points. GFPuv-HABP1 was also used for efficient fluorescence labeling of mineralized regions on the extracted human incisors. Our results demonstrate a simple and versatile application of inorganic-binding peptides conjugated with bioluminescence proteins as bi-functional bioimaging molecular probes that target mineralization, and which can be employed to a wide range of biomimetic processing and cell-free tissue engineering.     

Cytokinetic analysis of lung cancer by bromodeoxyuridine labeling of cytology specimens

Recent flow cytometric (FCM) studies have indicated the prognostic value of S-phase cells (SPF) in lung cancer. More refined cytokinetic analysis can be obtained by dual-parameter FCM, labeling S-phase cells with 5-bromodeoxyuridine (BrdUrd), which can be detected using a monoclonal antibody (MoAb) to BrdUrd. Tumor cells obtained through bronchoscopic brush were incubated for 1 hr in RPMI 1640 medium with 10% fetal calf serum and 10 microM BrdUrd. After fixation in ethanol, pepsin treatment, and DNA denaturation, the nuclei were stained with anti-BrdUrd MoAb and propidium iodide. From 14 of 20 patients, sufficient material was obtained (three adenocarcinoma and seven squamous cell, one giant cell, and three small cell carcinoma). The measured SPF ranged from 5.2% to 26%. The labeling index (LI), calculated as the ratio of the number of BrdUrd-labeled cells to the total number of aneuploid cells, or diploid cells in the case of a diploid tumor, ranged from 1.2% to 16.7%; LI and SPF correlated significantly (r = 0.69). In this study, we have demonstrated the feasibility of determining the actively DNA-synthesizing cells on brush material from lung cancer cells. In addition, some extra information can be obtained about the SPF population, including the fraction of unlabeled SPF, which could be of prognostic significance.     

In vitro RNA synthesis by intact rat brain nuclei

The characteristics of DNA-dependent RNA polymerase activity in intact rat cerebral cortex nuclei tested at low ionic strength are presented. The system was most dependent on the presence of spermidine and an ATP-generating system and to a lesser extent on Mg2+ and K+ for maximal incorporation. Substitution of Mg2+ by Mn2+ or of K+ by Na+ resulted in substantially less activity than under the optimum conditions described. Maximal incorporation was about 10 per cent that of brain nuclear systems of high ionic strength. In addition, the labelling patterns of the in vitro RNA products were shown to be very similar to those found in vivo. The stability of isolated nuclei toward degradation of RNA synthetic capacity and products formed was much greater than that of a similar liver system.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

Abstract. The present experiments with [¹⁴C]-thymidine (TdR) and [³H]-bromo-deoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 µg g body weight^-1 and that of BrdU is about 5·0 µg g body weight^-1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [³H]-TdR doses commonly used in cell kinetic studies this proportion is only 0-1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [³H]-TdR.     

In vitro assembly of multiple DNA fragments using successive hybridization

Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

The present experiments with [14C]-thymidine (TdR) and [3H]-bromodeoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 microgram g body weight-1 and that of BrdU is about 5.0 micrograms g body weight-1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [3H]-TdR doses commonly used in cell kinetic studies this proportion is only 0.1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [3H]-TdR.