Microtubules, organelle transport, and steroidogenesis in cultured adrenocortical tumor cells. 1. An ultrastructural analysis of cells in which basal and ACTH-induced steroidogenesis was inhibited by taxol

In adrenocortical cells, the first step in the enzymatic processing of cholesterol to steroid end products occurs in the mitochondria. ACTH increases mitochondrial cholesterol and steroidogenesis. In cultured mouse adrenocortical tumor cells, microtubule-based organelle motility may increase the proximity of mitochondria to the SER, lipid droplets and endoscome-derived lysosomes, thereby facilitating the transfer of cholesterol from these organelles to the mitochondrial outer membrane. ACTH may increase opportunities for the transfer by promoting organelle motility and by increasing the number of lysosomes. Taxol, a microtubule polymerizer, inhibits basal and ACTH-induced steroidogenesis in these cells, presumably at the step where mitochondria obtain cholesterol. We examined the ultrastructure of taxol-treated, unstimulated and ACTH-stimulated cells, seeking alterations which conceivably could interefer with the proposed organelle transport and encounters, and thus correlate with taxol's inhibition of steroidogenesis. Primary cultured cells were incubated in serum-containing medium for 4 hr with and without ACTH (10 mU/ml), with 10 micrograms/ml and 50 micrograms/ml of taxol, and with ACTH and taxol 10 or taxol 50 simultaneously. Culture media were analyzed for the presence of secreted steroids at the end of 1, 2, and 4 hr of incubation. At the end of the fourth hour, unstimulated cells and cells treated with ACTH, taxol 50, and both agents simultaneously, were fixed and processed for EM. Taxol inhibited basal and ACTH-induced steroidogenesis in a dose-dependent fashion. In both unstimulated and ACTH-stimulated cells, taxol 50 formed numerous microtubule bundles, but did not markedly change the distribution of mitochondria and lipid droplets. SER tubules, and clusters of Golgi fragments, endosomes, and lysosomes appeared to be translocated towards the cell periphery along some of the microtubules. Taxol permitted an ACTH-induced cell rounding and microfilament rearrangement considered to facilitate organelle motility. Our data indicate that taxol disrupts the formation of lysosomes by these adrenal cells, but it seemed unlikely that taxol's ultrastructural effects could prevent organelle transport proposed to cause meetings between mitochondria and the SER or lipid droplets, or prevent ACTH-caused increases in these encounters. Taxol may instead prevent the transfer of lipid droplet or SER-contained cholesterol to adjacent mitochondria, by a means not detectable in our electron micrographs.     

Effect of growth hormone and corticotropin on steroidogenesis in cultured rat adrenocortical cells

Primary cultures of rat adrenocortical cells responded to corticotropin (ACTH; 10 microU/ml) with peak steroid production within 24 h which declined thereafter. In the presence of ACTH and growth hormone (GH; 10 micrograms/ml), steroid production was significantly greater than with ACTH alone and was better maintained over several days. This latter response was not due to changes in cell number or multiplication and required several days to develop. GH also interacted with 10(-6) and 10(-5) M dibutyryl cyclic AMP (dbcAMP) to augment synthesis of corticosterone. At maximal doses of both ACTH and dbcAMP, GH did not have an additional effect on steroid production. In conclusion, GH has a stimulatory effect on steroid production when added in vitro but it is unlike the response seen in vivo in that it is less sensitive, additive rather than synergistic, and without effect on cell growth and multiplication.     

Cytoskeletal organization and cell organelle transport in basal epithelial cells of the freshwater sponge Spongilla lacustris

Summary Different antibodies against actin, tubulin and cytokeratin were utilized to demonstrate the spatial organization of the cytoskeleton in basal epithelial cells of the freshwater sponge Spongilla lacustris. Accordingly, actin is localized in a cortical layer beneath the plasma membrane and in distinct fibers within the cytoplasmic matrix. Microtubules exhibit a different distributional pattern by radiating from a perinuclear sheath and terminating at, the cell periphery; in contrast, intermediate filaments are lacking. Cytoplasmic streaming activity was studied by in-vivo staining of mitochondria and endoplasmic reticulum by means of fluorescent dyes. Single-frame analysis of such specimens revealed a regular shuttle movement of mitochondria and other small particles between the cell nucleus and the plasma membrane, which can be stopped in a reversible manner with the use of colcemid or colchicine but not with cytochalasin D. The results point to the microtubular system as a candidate for cell organelle transport, whereas the actomyosin system rather serves for changes in cellular shape and motility.     

Reversibility of ouabain induced inhibition of cell division and cation transport in Ehrlich ascites cells

Ouabain induced inhibition of cation transport and cell division in Ehrlich mouse ascites tumor cells is reversible, suggesting that this agent does not bind irreversibly to its site of action.     

The reversibility of the effects of ACTH and cytochalasin B on the ultrastructure and steroidogenic activity of adrenocortical tumor cells in vitro

We have demonstrated previously that the steroidogenic activity of ACTH on cultured adrenal tumor cells is associated with cell rounding and a rearrangement of microfilaments. Cytochalasin B (CB) also induces cell rounding, but changes the conformation of microfilaments and severely inhibits steroidogenesis. ACTH and CB may have different modes of action on the contractile machinery which are related to their opposing actions on steroidogenesis. To investigate this possibility further, we have examined the reversibility of the morphological and functional effects of these agents. Cultures were incubated for 1 hr, with and without ACTH (10 microU/ml of media), or with CB (50 micrograms/ml), or with both agents simultaneously. After a media wash, the cultures were incubated for 1 hr, with and without ACTH. The steroid production of the cells during pre- and post-washout incubations was determined, and some cultures were fixed for electron microscopy at the end of both incubation periods. The three- to ten-fold increases in steroidogenic activity of ACTH-stimulated cells declined during recovery incubations, but remained well above basal values. These cells nearly reflattened and began to regain stress fibers which had been 'pulled apart'. The 'washed out' ACTH-stimulated cells were often refractory to restimulation. Cells recovering from CB also reflattened. Masses of filamentous felt induced by the drug disappeared from the cytoplasm, lost microvilli reappeared and stress fibers reformed. The 20-50% inhibition of basal steroidogenesis by CB was completely reversed. When ex-CB-treated cells were incubated with ACTH, their morphology and steroid production were typical of acutely stimulated cells. The recovery behavior of cells incubated with ACTH and CB simultaneously reflected the observation that there were cell-specific responses to one agent or the other during initial incubations. The persistence of heightened steroidogenic activity following a washout of ACTH and the rapid reversal of the effects of CB strongly support the concept that regulated actomyosin interactions are an integral part of the steroidogenic process.     

Androgen-induced changes in Leydig cell ultrastructure and steroidogenesis in juvenile African catfish, Clarias gariepinus

The present report focuses on the mechanism(s) involved in the steroid-induced decrease of androgen production in immature African catfish testes that was observed in previous studies. Juvenile animals were implanted with Silastic pellets containing different 11-oxygenated androgens (11-ketotestosterone, KT; 11 beta-hydroxyandrostenedione, OHA; 11-ketoandrostenedione, KA), testosterone (T) or estradiol-17 beta (E2). Control groups received steroid-free pellets. Two weeks later, testis tissue fragments were either incubated with increasing concentrations of catfish luteinizing hormone (LH), or incubated with [3H]-pregnenolone ([3H]-P5) or [3H]-androstenedione ([3H]-A). Tissue fragments were also prepared for the quantitative assessment of Leydig cell morphology. Most of the parameters studied were not affected significantly by implantation of E2. Implantation of all androgens inhibited both the basal and the LH-stimulated androgen secretory capacity in vitro. This was associated with a reduced size of the Leydig cells and loss of half of their mitochondria. The studies on the metabolism of tritiated steroid hormones indicated that steroidogenic steps prior to 11 beta-hydroxylation, probably C17-20 lyase activity, were affected by all androgens. Although the effects of 11-oxygenated androgens and T on Leydig cells were mostly similar, previous work showed that only the 11-oxygenated androgens stimulated spermatogenesis, suggesting that distinct mechanisms of action are used by 11-oxygenated androgens and T. These mechanisms, however, seem to merge on the same target(s) to impair Leydig cell androgen production. Such a negative feedback mechanism may be of relevance in the context of the decline in androgen secretion per milligram testis tissue that accompanies the first wave of spermatogenesis in pubertal African catfish.     

Effects of lectins on cAMP production and steroidogenesis in cultured adrenal tumor cells

The plant lectins, concanavalin A (conA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA) stimulate steroidogenesis in cultured adrenal tumor cells. ConA maximally stimulated steroidogenesis at 100 μg/ml following an approximate 4 h lag phase. ConA stimulation was completely inhibited by α-methyl-d-mannopyranoside and the WGA effect was prevented by N-acetyl-d-glucosamine. It was also found that conA alone did not cause a measurable increase in either intra- or extracellular cyclic adenosine 3′5′-monophosphate (cAMP) production. In addition, conA when added simultaneously with adrenocorticotropin (ACTH) doubled the intra- and extracellular cAMP production over controls treated with ACTH alone. This enhancement effect was dose dependent. When Y-1 cells were preincubated with conA and then treated with either ACTH or cholera enterotoxin (CT) there was a dose- and time-dependent inhibition of induced cAMP production. In the case of CT, the inhibitory effect occurred even with simultaneous addition of conA and CT. This effect was reversed by addition of both α-methyl-d-mannopyranoside and washing with Eagle's minimal essential medium (MEM) 1 h after CT had bound to its receptor. This reversal was not apparent for the inhibitory effect of conA on ACTH-induced cAMP production which occurred after 2 h of preincubation with conA. These results demonstrate that conA, as well as the other plant lectins, interact with specific membrane receptors to reversibly stimulate steroid production as well as enhancing or inhibiting ligand-induced cAMP production in cultured adrenal tumor cells.     

Cinemicrographic observations of cultured adrenocortical tumor cells. Dynamic responses to ACTH and cytochalasin B

ACTH increases the basal steroidogenic activity of cultured adrenocortical tumor cells, whereas moderate-high doses of cytochalasin B (CB) inhibit both basal and ACTH-induced steroidogenesis. Previous ultrastructural studies have revealed that ACTH rearranges microfilaments in these adrenal cells, whereas CB causes microfilaments to aggregate into felt-like masses. It has been postulated that the ACTH effects may facilitate organelle motility and increase organelle interactions that are required for steroid biosynthesis, and that the CB-created "foci" may impede or prevent the organelle meetings. To shed light on these possibilities, we have employed 16 mm cinemicrography of unstimulated adrenal tumor cells and cells incubated for 1-2 h with ACTH (10 mU/ml), or low (10 micrograms/ml), or high (50 micrograms/ml) doses of CB. ACTH caused initial increases in membrane ruffling and a "flurry" of particle (organelle) activity above that seen in unstimulated cells. The stimulated cells then retracted from each other and began their characteristic "rounding up" in response to the hormone. Particles appeared to move towards the nucleus, and in fully-rounded cells were extremely congested. Steroid production rose several fold above basal levels. CB10 produced slight-marked cell convexities, nearly stopped particle motility and inhibited steroid production moderately. CB50 produced an asymmetrical, spidery cell form, stopped membrane ruffling and particle motility and abolished steroidogenesis. After a washout of CB50, particle motility resumed nearly immediately. Our CB data indicate that associations between particles, presumably between mitochondria and various sources of cholesterol, are prerequisite for basal steroidogenesis in the adrenal tumor cells. In ACTH-stimulated cells, increases in steroid output correspond with increased opportunities for particle associations. These opportunities appear to arise directly or indirectly from ACTH effects on microfilaments. The responses of microfilaments to the hormone may be particularly intense in tumorous forms. By these means, the cells may express their differentiated function, although their cytoplasm has a distinctly unspecialized appearance.     

Immunocytochemistry of the Microtubules of fat-laden cells. Brown fat cells and adrenocortical cells in primary monolayer culture

Cytoplasmic microtubules of fat-laden cells containing fine lipid droplets, such as brown fat cells and adrenocortical cells, were studied in relation to the metabolism of intracellular lipid. In these cells, the amount and distribution of lipid droplets reflect the state of inherent cellular function. Materials used were primary monolayer culture of fetal rat brown fat cells and that of bovine adrenocortical cells. The method was the immunocytochemistry with anti-tubulin antibody. When brown fat cells were being lipolyzed or the steroidogenesis of adrenocortical cells were being stimulated, the cytoplasmic microtubules in the cells were organized in a radial pattern in response to the behavior of the lipid droplets. It is assumed that the microtubules were in the regulation of cellular function in terms of the metabolism of lipid droplets in these cells. We have devised, in the course of the current study, a double fluorescence technique as an observational method whereby microtubules were observed immunocytochemically and lipid droplets by a secondary fluorescence with phosphine E staining.     

Sequential study on the influence of adriamycin on cell proliferation and ultrastructure of cultured mammary tumor cells

The time-dependent cytocidal and growth inhibitory effects of Adriamycin (ADM) on monolayer cultures of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor cells were analyzed. The inhibitory effect on cell proliferation was assessed by colony formation in soft agar. Growth inhibition and [3H]thymidine labeling indices clearly demonstrate a dose-dependent antimitotic and cytotoxic effect of the drug. At low concentrations (10(-9)-10(-8) M), 90-100% of cells survived 24-hr exposure. At a higher concentration (10(-5) M), 75-80% of cells survived after 8-hr exposure; by 72 hr only 20-30% of the cells remained. Autoradiographic examination of the pulse-labeled cultures demonstrated no change in the proportion of cells in S-phase during the first 4 hr of treatment. Subsequently DNA synthesis was completely abolished and remained inhibited for the duration of the experiment (72 hr). Clonogenic assay revealed a complete arrest of growth in cells exposed to 10(-5) M ADM and greater than 60% inhibition of cell proliferation at 10(-7) M. Ultrastructural changes were not observed in cells during the first 4 hr of treatment; however, after 8 hr most surviving cells exhibited alterations in nuclear chromatin. The surviving cells showed mitochondrial degeneration, myelin body formation, and vacuolization of the endoplasmic reticulum. This study shows the potential usefulness of the primary culture system in drug evaluation. In addition, serial observation of the effects of ADM revealed a cell subpopulation of the primary culture with differential sensitivity to the drug.     

Contact inhibition, polyribosomes, and cell surface membranes in cultured mammalian cells

An “overlay” method for rapidly and synchronously inducing contact inhibition in normal cultured cells has been developed. Using this method, disaggregation of cytoplasmic polyribosomes has been observed to occur within a matter of hours after overlay, followed by a decrease in cellular ribosomal RNA. Polysome disaggregation was influenced by the extent of cell-cell interaction and was inhibited by pretreatment of overlay cells with cycloheximide. Treatment of underlay cells with cytosine arabinoside also induced polysome disaggregation, but only after an appreciable lag as compared to that observed in overlaid cultures. Disaggregation could be induced by this method in cultured cells derived from normal tissue but not in cells derived from cancerous tissue. Polysome synthesis in growing “normal” cells (as measured by incorporation of tracer uridine into RNA) was markedly decreased when a cell surface membrane preparation was added to cultures.     

Effects of a water-soluble extract of Cordyceps sinensis on steroidogenesis and capsular morphology of lipid droplets in cultured rat adrenocortical cells

Cordyceps sinensiscontains a factor that stimulates corticosteroid production in the animal model. However, it is not known whether this drug acts directly on the adrenal glands or indirectly via the hypothalamus-pituitary axis. In the present study, we used primary rat adrenal cell cultures to investigate the pharmacological function of a water-soluble extract of Cordyceps sinensis(CS) and thesignaling pathway involved. Radioimmunoassay of corticosterone indicated that the amount of corticosterone produced by adrenal cells is increased in a positively dose-dependent manner by CS, reaching a maximun at 25 μg/ml. This stimulating effect was seen 1 h after CS treatment and was maintained for up to 24 h. Concomitantly, the lipid droplets in these cells became small and fewer in number. Immunostaining with a monoclonal antibody, A2, a specific marker for the lipid droplet capsule, demonstrated that detachment of the capsule from the lipid droplet occurs in response to CS application and that the period required for decapsulation is inversely related to the concentration of CS applied. The mechanism of CS-induced steroidogenesis is apparently different from that for ACTH, since intracellular cAMP levels were not increased in CS-treated cells. However, combined application with calphostin C, a PKC inhibitor, completely blocked the effect of CS on steroidogenesis, suggesting that activation of PKC may be responsible for the CS-induced steroidogenesis. J. Cell. Biochem. 69:483–489, 1998. © 1998 Wiley-Liss, Inc.     

Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrenal cells of vitamin E-deficient rats.

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5'-triphosphate [14C]ATP to cyclic [14C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [14C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induced cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [14C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [14C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [14C]AMP continued to be formed. The in vitro addition of alpha-tocopherol reduced the inhibition of ACTH-induced cyclic [14C]AMP formation by ascorbate in Vitamin E-deficient rats. These studies suggest that alpha-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with the membrane-bound enzyme adenylate cyclase.     

Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle.

We examined the intracellular transport of sterol in living cells using a naturally fluorescent cholesterol analog, dehydroergosterol (DHE), which has been shown to mimic many of the properties of cholesterol. By using DHE loaded on methyl-beta-cyclodextrin, we followed this cholesterol analog in pulse-chase studies. At steady state, DHE co-localizes extensively with transferrin (Tf), a marker for the endocytic recycling compartment (ERC), and redistributes with Tf in cells with altered ERC morphology. Expression of a dominant-negative mutation of an ERC-associated protein, mRme-1 (G429R), results in the slowing of both DHE and Tf receptor return to the cell surface. [3H]Cholesterol is found in the same fraction as 125I-Tf on sucrose density gradients, and this fraction can be specifically shifted to a higher density based on the presence of horseradish peroxidase-conjugated Tf in the same organelle. Whereas vesicular transport of Tf and efflux of DHE from the ERC are entirely blocked in energy-depleted cells, delivery of DHE to the ERC from the plasma membrane is only slightly affected. Biochemical studies performed using [3H]cholesterol show that the energy dependence of cholesterol transport to and from the ERC is similar to DHE transport. We propose that a large portion of intracellular cholesterol is localized in the ERC, and this pool might be important in maintaining cellular cholesterol homeostasis.     

Effects of cytochalasin D on the distribution of actin and ACTH-induced steroidogenesis in cultured embryonic adrenal gland cells from the Pekin duck (Anas platyrhynchos)

Cultured steroidogenic cells derived from the adrenal glands of duck embryos were used to study changes in the distribution of actin associated with the corticotropic responsiveness. Actin-containing components were identified by rhodamine-phalloidin staining. The actin in most of the unstimulated cells occurred as stress fibers that either ran parallel throughout the cell or were present as domains of parallel fibers at angles to one another. When incubated in Krebs-Henseleit buffer containing 1–24 ACTH, the cells released approximately equal amounts of corticosterone and aldosterone. Incubation of the cells in buffer containing cytochalasin D caused the cells to lose their stress fibers, and the actin became distributed at the periphery in what appeared to be fragments of stress fibers and clumps of fibrous material in the central cytoplasm. Although cytochalasin D did not affect the basal output of corticosterone and aldosterone, the 1–24 ACTH-induced rates of both hormones were suppressed significantly. After the cells had been washed in unadulterated buffer, the normal distribution of actin stress fibers was restored and the cells responded normally when incubated in buffer containing 1–24 ACTH. These results suggest that the actin components of the cytoskeleton are important determinants of corticotropin-induced steroidogenic responsiveness.     

Studies on lipoprotein and adrenal steroidogenesis: I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human adrenocortical cells

The roles of human low density lipoprotein (LDL)- cholesterol and high density lipoprotein (HDL)- cholesterol on adrenal steroidogenesis were investigated using cultured human adult and fetal adrenocortical cells and the findings were then compared to those obtained with bovine adrenocortical cells. The secretion of cortisol in both human and bovine adrenocortical cells was dose-dependently increased by the administration of LDL- or HDL-cholesterol in the presence of adrenocorticotropin (ACTH). LDL-cholesterol was utilized to a greater extent than HDL-cholesterol in both human and bovine adrenal steroidogenesis in the presence of ACTH. Exogenous lipoprotein-derived cholesterol was less utilized in human adrenal steroidogenesis than in bovine adrenal steroidogenesis, compared to the endogenous cholesterol. An increase in the secretion of cortisol and dehydroepi androsterone sulfate (DHEA-S) continued for the 5-day culture period, in the presence of lipoprotein cholesterol and ACTH in both human adult and fetal adrenocortical cells. The secretion of aldosterone increased on the first day of the culture period, then gradually decreased for the 5-day culture period in human adult adrenocortical cells, but not in human fetal adrenocortical cells in the presence of lipoprotein cholesterol and ACTH. These findings demonstrate that exogenous cholesterol utilized in the biosynthesis of steroids is mainly from LDL-cholesterol in both human adult and fetal adrenals and bovine adrenal and the proportion of cholesterol synthesized de novo is significantly larger in the human adult adrenal than in the bovine adrenal.     

Reversibility of ultrastructural, contractile function and Ca2+ transport changes in guinea pig hearts after global ischemia

To understand the subcellular basis of contractile failure due to ischemia-reperfusion injury, effects of 20, 60, and 90 min of global ischemia followed by 30 min of reperfusion were examined in isolated guinea pig hearts. Cardiac ultrastructure and function as well as Ca2+ transport abilities of both mitochondrial and microsomal fractions were determined in control, ischemic, and reperfused hearts. Hearts were unable to generate any contractile force after 20 min of ischemia and showed a 75% recovery upon reperfusion. However, there were no significant changes in the subcellular Ca2+ transport in the 20-min ischemic or reperfused hearts. When hearts were made ischemic for 60 and 90 min, the recovery of contractile force on reperfusion was 50 and 7%, respectively. There was a progressive decrease in mitochondrial and microsomal Ca2+ binding and uptake activities after 60 and 90 min of ischemia; these changes were evident at various times of incubation period and at different concentrations of Ca2+. Mitochondrial Ca2+ transport changes were only partially reversible upon reperfusion after 60 and 90 min of ischemia, whereas the microsomal Ca2+ binding, uptake and Ca2+ ATPase activities deteriorated further upon reperfusion of the 90-min ischemic hearts. Ultrastructural changes increased with the duration of the ischemic insult and reperfusion injury was extensive in the 90-min ischemic hearts. These data show that the lack of recovery of contractile function upon reperfusion after a prolonged ischemic insult was accompanied by defects in sarcoplasmic reticulum Ca2+ transporting properties and structural damage.     

Presence of human papillomavirus genome in human tumor cell lines and cultured cells.

A series of 47 human carcinoma cell lines and their cultured cells were examined for human papillomavirus (HPV) genomes with the use of an HPV detection kit (DNA-RNA hybridization, mixed HPV DNA probe of types 6, 11, 16, 18, 31, 33 and 35). Four of 8 cases of mild dysplasia, 3 of 9 cases of severe dysplasia, 3 of 7 cases of carcinoma in situ, 3 of 15 cases of uterine carcinoma and 5 of 6 cases of condyloma acuminatum were shown to contain the HPV DNA genome in primary cultured cells, while HPV was not detected in the third-passage cells except for the three cases of large cell, nonkeratinizing squamous cell carcinoma. HPV was also not detected in such normal tissues as uterine cervical squamous epithelium, uterine cervical columnar epithelium and endometrium. The presence of HPV DNA genomes was detected consistently in the passages of three lines (SKG-II, HKMUS and HKTUS; large cell nonkeratinizing squamous cell carcinomas of the uterine cervix) with the use of the Southern Blot method (DNA-DNA hybridization, mixed HPV probe of types 6, 11, 16 and 18). HPV type 16 DNA was detected in HKTUS, and HPV type 18 DNA was found in SKG-II and HKMUS. The other 44 cell lines, including ovarian carcinoma, endometrial carcinoma, sarcoma, gastric cancer, pancreatic cancer and rectal cancer, were negative for the HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35 genomes under stringent hybridization conditions.