Phyllis M. Novikoff

DIFFUSION ARTIFACTS IN 3,3'-DIAMINOBENZIDINE CYTOCHEMISTRY

throughout the cytoplasm (Figs. 2 and 3). The reaction product was seen as coarse masses of granules or accumulation of fine threads forming a mesh. The albumin-containing cells were scattered diffusely throughout the lobules. Most of cells situated in the centrilobular area contained a large amount of albumin. The control sections showed no staining. The localization of albumin in liver tissue observed is similar to that reported by using an immunofluorescent antibody method (5). In the lymph nodes of rabbits hyperimmunized with HPO, the cells containing antibody against HPO were mainly localized in the medullary cords adjacent to the lumen of the lymph sinuses or in the lumen itself (Fig. 4). The antibody-containing cells were classified into two types (Fig. 5). One is plasma cell, which had an eccentrically situated nucleus and contained a large amount of antibody throughout the cytoplasm. The other cell type contained only a small amount of antibody in the thin rim of cytoplasm. These cells, round in shape and small in size, may be interpreted as lymphoblasts or lymphoplasmacytes (2). In the intercellular space, there were often small granules of the reaction product. Since the reaction product was observed only occasionally in the intercellular space in sections not exposed to HPO prior to incubation with substrate for peroxidase, most of intercellular reaction product was thought to indicate the localization of antibody in this space. There were few antibody-containing cells in the germinal center. These results are also identical with those reported with frozen or paraffin sections (9). Our results indicate that fixation, dehydration and embedding employed do not disturb the antigen-antibody reaction. Furthermore, GMA sect ions gave clearer localization of the antigen and antibody at both the cytologic and histologic level than frozen or paraffin sections. Several tissue antigens were localized immunocytochemically by Nakane (8) by using methacrylate-embedded or Epon-embedded ultrathin sections and by partially removing the embedding medium. The embedding medium is difficult to remove from thin sections, however. The use of GMA as embedding medium is expected to simplify the procedure because of the dispensability of the removal of embedding medium. REFERENCES

MRP3, a new ATP-binding cassette protein localized to the canalicular domain of the hepatocyte

Bile secretion in liver is driven in large part by ATP-binding cassette (ABC)-type proteins that reside in the canalicular membrane and effect ATP-dependent transport of bile acids, phospholipids, and non-bile acid organic anions. Canalicular ABC-type proteins can be classified into two subfamilies based on membrane topology and sequence identity: MDR1, MDR3, and SPGP resemble the multidrug resistance (MDR) P-glycoprotein, whereas MRP2 is similar in structure and sequence to the multidrug resistance protein MRP1 and transports similar substrates. We now report the isolation of the rMRP3 gene from rat liver, which codes for a protein 1522 amino acids in length that exhibits extensive sequence similarity with MRP1 and MRP2. Northern blot analyses indicate that rMRP3 is expressed in lung and intestine of Sprague-Dawley rats as well as in liver of Eisai hyperbilirubinemic rats and TR- mutant rats, which are deficient in MRP2 expression. rMRP3 expression is also transiently induced in liver shortly after birth and during obstructive cholestasis. Antibodies raised against MRP3 recognize a polypeptide of 190-200 kDa, which is reduced in size to 155-165 kDa after treatment with endoglycosidases. Immunoblot analysis and immunoconfocal microscopy indicate that rMRP3 is present in the canalicular membrane, suggesting that it may play a role in bile formation.

Paclitaxel shows cytotoxic activity in human hepatocellular carcinoma cell lines

Paclitaxel stabilizes microtubules with inhibition of mitotic spindle formation and has been found effective in several solid cancers. To test whether paclitaxel could be cytotoxic in human HCC cell lines, we used established HuH-7 and HepG2 cell lines. Changes in cell number, DNA synthesis rates and cell viability were determined. We tested whether paclitaxel-treated cells underwent apoptosis, microtubular reorganization, and cell cycle restriction. Studies also examined whether chemosensitization with verapamil enhanced the antitumor activity of paclitaxel. The cell viability was impaired at greater than 0.01 microM paclitaxel concentrations (LD50, 0.8 microM), with flow cytometry indicating accumulation of cells in G2/M, and immunostaining showing polymerized microtubules with characteristic banding patterns. This G2/M restriction was further characterized by flow cytometry, which revealed cyclin A and cdc2 kinase accumulation in paclitaxel-treated cells. Exposure to paclitaxel decreased [3H]thymidine incorporation into DNA in cells at 24 h but this significantly increased at 72 h, most likely due to DNA repair mechanisms related to cell cycle restriction. The cell death was via both apoptotic and non-apoptotic mechanisms. Finally, co-administration of the chemosensitizer verapamil in doses as little as 1 microM increased the antitumor efficacy of paclitaxel by up to five-fold and changed the LD50 of paclitaxel to 0.1 microM. The findings indicate that paclitaxel is cytotoxic to cultured hepatocellular carcinoma cells. Clinical studies of paclitaxel in patients with hepatocellular carcinoma may help determine additional therapies.

MICROPEROXISOMES AND PEROXISOMES IN RELATION TO LIPID METABOLISM

We will begin with the last topic in the conference program abstract of this paper: what we call constellations. These constellations include: (a) “cytosolic” lipid spheres (in the sense that the spheres are not enclosed by membranes visible by conventional electron microscopy-whatever their relation might be to the “cytoskeleton” of Keith Porter’ or the “cytosol” of Sheldon Penman’); (b) endoplasmic reticulum or ER; (c ) peroxisomes (in rat hepatocytes, in proximal renal tubules, and in a few other cell types) or microperoxisomes (in all animal cell types studied and in some plant cells as well); and (d) mitochondria. These constellations show such intimate cytological association that they must have functional significance. But electron microscopy, even when supplemented with cytochemically valid reactions for enzyme localizations [for a list of those we use in our laboratory, see ref. 3) can only be qualitative. Such methods could describe the presence and distributions of peroxisomes [also of lysosomes) among the various cell types and tissues. With cytochemistry, we were able to describe particles hitherto not known to exist. And so, Christian de Duve’s brilliant PANEL DISCUSSION in the last New York Academy of Sciences Conference held in 196g4 needs emendation regarding microperoxisomes. In the present Conference‘ we have lost the word “Microbodies.“ and have gained the word “International.“ A good bargain, yes? Yet, cytochernistry cannot yield quantitative data such as obtained by biochemists and molecular biologists. Nor can it establish the concept of peroxisome or the concept of lysosome. Each concept involves a collection of enzymes, quantitatively assayed, within a distinctive cytoplasmic particle. On the other hand, in 1979, P. Lazarow et al.,& from their extensive gel analyses. should not have drawn the conclusion that the peroxisome membrane and ER are not continuous. Paul was unable to attend the meeting and thus Ann Hubbard had to refute our comment to this effect, which we read at the meeting. During our comment we projected FIGURES 10 and 11 of the present publication. The most recent review of the peroxisome-glyoxysome field is that of N. E. T01bert.~ The constellations are shown diagrammatically in FIGURE 1, an unpublished diagram. and in two micrographs, one from a rat hepatocyte’ (FIG. 2) and another

Dichotomous development of the organic anion transport protein in liver and choroid plexus

Both adult liver and choroid plexus express the organic anion transport protein (oatp1) and transport [35S]bromosulfophthalein (BSP). Studies of the developing rat liver reveal that oatp1 mRNA and protein do not begin to be expressed until 15 days postnatal and are at adult levels by 30 days. Uptake of [35S]BSP follows the same time course. In contrast, neonatal rat choroid plexus expresses oatp1 mRNA and protein. When quantified on a weight basis, the uptake of [35S]BSP in choroid plexus is lower in the adult than at earlier stages of development. Although fluorescence confocal microscopy of adult rat choroid plexus shows that oatp is localized to the apical surface, facing the cerebrospinal fluid, this method reveals an intracellular localization of oatp1 in the neonate. Approximately 12 wk are required for the appearance of the adult pattern of distribution. Changes in the localization and activity of oatp1 during development could play an important role in the pathobiology of maturation of the liver and the central nervous system.Both adult liver and choroid plexus express the organic anion transport protein (oatp1) and transport [35S]bromosulfophthalein (BSP). Studies of the developing rat liver reveal that oatp1 mRNA and protein do not begin to be expressed until 15 days postnatal and are at adult levels by 30 days. Uptake of [35S]BSP follows the same time course. In contrast, neonatal rat choroid plexus expresses oatp1 mRNA and protein. When quantified on a weight basis, the uptake of [35S]BSP in choroid plexus is lower in the adult than at earlier stages of development. Although fluorescence confocal microscopy of adult rat choroid plexus shows that oatp is localized to the apical surface, facing the cerebrospinal fluid, this method reveals an intracellular localization of oatp1 in the neonate. Approximately 12 wk are required for the appearance of the adult pattern of distribution. Changes in the localization and activity of oatp1 during development could play an important role in the pathobiology of maturation of the liver and the central nervous system.

The cytochemical demonstration of GERL in rat hepatocytes during lipoprotein mobilization.

When a semisynthetic diet containing 1% orotic acid (OA) is fed to rats, the endoplasmic reticulum (ER) of hepatocytes vesiculates and lipoprotein (LP) droplets accumulate within the vesicles. When clofibrate (ethyl chlorophenoxyisobutyrate, CPIB) is added to the orotic acid-rich diet, the ER cisternae reform and the LP is mobilized through the reconstituted ER. A remarkable restoration of normal hepatocyte ultrastructure occurs except for a few organelles. From their morphological appearance it was suggested that cisternae which became dilated with small LP particles were part of GERL, abnormally enlarged. The present communication validates this interpretation through ultrastructural cytochemistry which can distinguish GERL from the adjacent Colgi apparatus. GERL shows acid phosphatase (AcPase) but not thiamine pyrophosphatase (TPPase) activity. In contrast, the adjacent Golgi element shows thiamine pyrophosphatase but not acid phosphatase activity. From such cytochemical studies we have recently proposed that GERL in normal rat hepatocytes may be involved in transforming LP particles, by enzymes like lipases that were presumed to be present in this hydrolase-rich portion of smooth ER. In the situation studied in this communication, the addition of ethyl chlorophenoxyisobutyrate to the diet causes the release from the ER of large amounts of LP to the Golgi apparatus and to GERL. Apparently the capacity of GERL to metabolize LP is exceeded and lipid accumulates in the residual bodies.

Stem Cells and Rat Liver Carcinogenesis: Contributions of Confocal and Electron Microscopy

Microscopic analysis in combination with cytochemistry and immunocytochemistry has revealed the presence of four cell types not previously described in the portal area and parenchyma of the liver from an experimental rodent hepatocarcinogenic rat model. Within the intrahepatic bile ductules, which proliferate after administration of chemical carcinogens and partial hepatectomy, small, undifferentiated nonpolarized, nonepithelial cells with a blast-like phenotype and polarized epithelial cells different from the polarized epithelial cells that typically line the walls of the bile ductules were found. In the connective tissue stroma surrounding the bile ductules, nonpolarized epithelial cells with hepatocyte phenotype were found. In the parenchyma, subpopulations of bile ductule epithelial cells that established ATPase-positive bile canalicular structures, including the formation of desmosomes and tight junctions, with parenchymal hepatocytes within the hepatic lobule were found. These observations raise the following questions in this model. Are there undifferentiated progenitor cells with stem cell-like properties within bile ductules? What are the interrelations of the newly described cell types with each other, with parenchymal hepatocytes, with preneoplastic nodules, and with hepatomas? Do the heterogeneous cell types within the bile ductules, in the surrounding connective tissue, and within the hepatic cords represent intermediate stages of single or multiple cell lineage pathways leading to hepatocyte differentiation, liver regeneration, and/or preneoplastic nodule formation?

Albumin and Collagen mRNA Expression in Normal and Analbuminemic Rodent Liver: Analysis by In Situ Hybridization Using Biotinylated Probes'

We used in situ nucleic acid hybridization cytochemistry to examine cell types and subcellular sites expressing albumin (alb) or pro alpha 2 collagen (col) mRNA in livers from normal and analbuminemic rodents. Biotinylated cDNA or RNA probes were applied to aldehyde-fixed, non-frozen sections and the resulting DNA-RNA or RNA-RNA hybrids were subsequently visualized by enzymatic detection of either peroxidase or alkaline phosphatase conjugated to anti-biotin IgG or streptavidin. In normal rat liver, alb mRNA was expressed in all hepatocytes and was localized to discrete subcellular structures distributed as aggregates in the cytoplasm and in specific structures encircling the nucleus; these subcellular structures most likely represent the endoplasmic reticulum and nuclear envelope. In mouse liver, pro alpha 2 col mRNA was identified in a subpopulation of sinusoidal lining cells which have the morphological appearance of lipocytes. In liver from analbuminemic rats, a small number of hepatocytes, distributed throughout the hepatic lobule, expressed alb mRNA at high levels; the subcellular distribution of this alb mRNA was essentially identical to that observed in normal rat hepatocytes. Since non-radioactive in situ hybridization detected mRNA within the boundaries of individual cells and showed its precise subcellular location under conditions in which there was excellent preservation of tissue morphology, this procedure should be useful for a wide variety of histopathologic studies.

Cytochemical Staining Reactions for Enzymes in Cytoplasmic Organelles

Recent reports on the use of lead precipitating procedures for localizing intracellular phosphatases and criticism of these procedures (see (1) and (2) for the most recent interchange of views) have left undiminished the value of using the following phosphatases as cytochemical “markers” for light and electron microscopy (3, 4): plasma membrane — nucleoside phosphatases (e.g., Mn++ — and Mg++-stimulated “ATPase”, 5’-nucleotidase, Co++-stimulated CMPase, etc.); endoplasmic reticulum (ER) of liver, kidney, endocrine-secreting cells, etc. -- a nucleoside diphosphatase (NDPase) that hydrolyzes IDP, UDP, GDP (also TPP) but not CDP or ADP (5, 6); Golgi apparatus -- NDPase, thiamine pyrophosphatase (TTPase) (see 6)); Golgi-endoplasmic reticulumlysosome (GERL) -- acid phosphatase; and lysosomes -- acid phosphatase. Optimal oxidation at high pH and relatively high H2O2 concentration of diaminobenzidine may be used to “mark” peroxisomes because of their high catalase content (7, 8). Mitochondria oxidize diaminobenzidine optimally at low pH and low H2O2 concentration (7).

Human liver cell trafficking mutants: characterization and whole exome sequencing.

The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α’’. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.