Natural history of human papillomavirus infection in non-vaccinated young males: low clearance probability in high-risk genotypes.

In this study, we aimed to investigate the clearance of type-specific genital human papillomavirus (HPV) infection in heterosexual, non-HPV-vaccinated males whose female partners were positive to HPV DNA tests. All consecutive men attending the same sexually transmitted diseases (STD) centre between January 2005 and December 2006 were considered for this study. All subjects (n = 1009) underwent a urologic visit and microbiological tests on first void, midstream urine and total ejaculate samples. One hundred and five patients were positive for HPV DNA (10.4 %; mean age: 34.8 ± 5.8 years) and consented to clinical examination and molecular diagnostic assays for HPV detection scheduled every 6 months (median surveillance period of 53.2 months). HPV genotypes were classified as high risk, probable high risk and low risk. HPV-positive samples which did not hybridise with any of the type-specific probes were referred to as positive non-genotypeable. At enrollment, the distribution of HPV genotypes was as follows: high-risk HPV (n = 37), probable high-risk HPV (n = 6), low-risk HPV (n = 23) and non-genotypeable HPV (n = 39). A high HPV genotype concordance between stable sexual partners emerged (kappa = 0.92; p < 0.001). At the end of the study, 71/105 (67.6 %) subjects were negative for HPV (mean virus clearance time: 24.3 months). With regard to the HPV genotype, virus clearance was observed in 14/37 (37.8 %) high-risk HPV cases, 6/6 (100 %) probable high-risk HPV cases, 20/23 (86.9 %) low-risk HPV cases and 31/39 (79.5 %) non-genotypeable cases. The high-risk HPV genotypes showed the lowest rate and probability of viral clearance (p < 0.001). In our series, high-risk HPV infections were more likely to persist over time when compared with other HPV genotypes.

RENEB Inter-Laboratory Comparison 2021: The Cytokinesis-Block Micronucleus Assay.

The goal of the RENEB inter-laboratory comparison 2021 exercise was to simulate a large-scale radiation accident involving a network of biodosimetry labs. Labs were required to perform their analyses using different biodosimetric assays in triage mode scoring and to rapidly report estimated radiation doses to the organizing institution. This article reports the results obtained with the cytokinesis-block micronucleus assay. Three test samples were exposed to blinded doses of 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 13 mA, ∼75 keV, 1 Gy/min). These doses belong to 3 triage categories of clinical relevance: a low dose category, for no exposure or exposures inferior to 1 Gy, requiring no direct treatment of subjects; a medium dose category, with doses ranging from 1 to 2 Gy, and a high dose category, after exposure to doses higher than 2 Gy, with the two latter requiring increasing medical attention. After irradiation the test samples (no. 1, no. 2 and no. 3) were sent by the organizing laboratory to 14 centers participating in the micronucleus assay exercise. Laboratories were asked to setup micronucleus cultures and to perform the micronucleus assay in triage mode, scoring 500 binucleated cells manually, or 1,000 binucleated cells in automated/semi-automated mode. One laboratory received no blood samples, but scored pictures from another lab. Based on their calibration curves, laboratories had to provide estimates of the administered doses. The accuracy of the reported dose estimates was further analyzed by the micronucleus assay lead. The micronucleus assay allowed classification of samples in the corresponding clinical triage categories (low, medium, high dose category) in 88% of cases (manual scoring, 88%; semi-automated scoring, 100%; automated scoring, 73%). Agreement between scoring laboratories, assessed by calculating the Fleiss' kappa, was excellent (100%) for semi-automated scoring, good (83%) for manual scoring and poor (53%) for fully automated scoring. Correct classification into triage scoring dose intervals (reference dose ±0.5 Gy for doses ≤2.5 Gy, or reference dose ±1 Gy for doses >2.5 Gy), recommended for triage biodosimetry, was obtained in 79% of cases (manual scoring, 73%; semi-automated scoring, 100%; automated scoring, 67%). The percentage of dose estimates whose 95% confidence intervals included the reference dose was 58% (manual scoring, 48%; semiautomated scoring, 72%; automated scoring, 60%). For the irradiated samples no. 2 and no. 3, a systematic shift towards higher dose estimations was observed. This was also noticed with the other cytogenetic assays in this intercomparison exercise. Accuracy of the rapid triage modality could be maintained when the number of manually scored cells was scaled down to 200 binucleated cells. In conclusion, the micronucleus assay, preferably performed in a semi-automated or manual scoring mode, is a reliable technique to perform rapid biodosimetry analysis in large-scale radiation emergencies.

Eradication of Chlamydia trachomatis parallels symptom regression in chronic bacterial prostatitis patients treated with a fluoroquinolone-macrolide combination.

To investigate the association between eradication of Chlamydia trachomatis (CT) and symptom regression in chronic prostatitis, 55 symptomatic patients were subjected to segmented tests to localise CT in first voided urine (VB1), prostatic secretions (EPS), post-massage voided (VB3) or semen specimens. Patients were divided in three treatment groups: the 'urethral involvement' group ('U': VB1 positive, EPS/VB3/Semen negative) was treated with 500 mg day(-1) azithromycin for 3 days. The 'prostatitis' group ('P': VB1 negative, EPS/VB3/semen positive) with 4-week levofloxacin-azithromycin combination. A third group, 'U+P' (VB1, EPS/VB3/semen positive) received both treatments in sequence. In P patients, eradication of CT was paralleled by marked, sustained symptom improvement and by significant decrease of serum prostate-specific antigen (PSA) levels. Compared with U patients, undergoing rapid regression of symptoms related to painful micturition after short-term azithromycin, U+P patients showed symptom and pathogen persistence in VB3/EPS/semen and required additional treatment with 4-week levofloxacin-azithromycin to achieve pathogen eradication, symptom regression, and decrease of PSA. Our results support a causative role of CT in chronic bacterial prostatitis. In the presence of a positive urethral localisation of the pathogen, thorough microbiological investigation together with focused symptom analysis may reveal an underlying chlamydial prostatitis and direct effective therapy with appropriate antibacterial agents.

Characterization of the c.190T>C missense mutation in BRCA1 codon 64 (Cys64Arg).

In the Milan area (Northern Italy), we identified a family characterized by a high prevalence of ovarian and breast cancer cases (5 out of 6 subjects, over 3 generations), and a predominant prevalence of ovarian lesions (4 out of 5 patients). Analysis of BRCA1 and BRCA2 genes allowed the identification of the missense c.190T>C mutation in codon 64 (Cys64Arg) of BRCA1. The aims of the present investigation were to characterize the functional implications of the c.190T>C mutation at the molecular level, and to search whether additional polymorphisms might be linked to the peculiar phenotypic features observed in the Italian pedigree. Molecular modelling studies suggested that substitution of the cysteine 64 with an arginine likely disrupts the architecture of the BRCA1 RING finger domain, responsible for the interaction with BARD1, essential for the tumor-suppressor activity of the BRCA1-BARD1 complex. By splicing site information analysis, exonic splicing enhancer site characterization, and analysis of transcript fragment length and sequence, we showed that the c.190T>C mutation was able to modulate the splicing of exon 5 in a fashion opposite to the c.190T>G transversion, responsible for the functionally-related Cys64Gly amino acid substitution. Genotyping of BRCA1 and BRCA2 in the Italian family revealed the presence of two significant polymorphisms: the cancer-associated c.2612C>T SNP in BRCA1, and the c.-26G>A SNP in the BRCA2 gene, acting as an ovarian cancer risk modifier in carriers of deleterious BRCA1 mutations. Analysis of these SNPs in a genotypically-unrelated Polish family, characterized by prevalent breast neoplasms in carriers of the c.190T>C mutation, revealed a genetic profile consistent with the hypothetic role of both polymorphisms.

Role in anxiety behavior of the endocannabinoid system in the prefrontal cortex.

In the present study we explored with a multidisciplinary approach, the role of anandamide (AEA) in the modulation of anxiety behavior at the level of the prefrontal cortex (PFC). Low doses of the metabolically stable AEA analog, methanandamide, microinjected into the PFC, produced an anxiolytic-like response in rats, whereas higher doses induced anxiety-like behaviors. Pretreatment with the selective antagonist of CB1 or TRPV1 receptors (AM251 and capsazepine, respectively) suggested that the anxiolytic effect evoked by AEA might be due to the interaction with the CB1 cannabinoid receptor, whereas vanilloid receptors seem to be involved in AEA anxiogenic action. When AEA contents in the PFC were increased by microinjecting the selective inhibitor of fatty acid amide hydrolase (FAAH), URB597, we observed an anxiolytic response only at low doses of the compound and no effect or even an anxiogenic profile at higher doses. In line with this, a marked decrease of AEA levels in the PFC, achieved by lentivirus-mediated local overexpression of FAAH, produced an anxiogenic response. These findings support an anxiolytic role for physiological increases in AEA in the PFC, whereas more marked increases or decreases of this endocannabinoid might lead to an anxiogenic response due to TRPV1 stimulation or the lack of CB1 activation, respectively.

5-Lipoxygenase and anandamide hydrolase (FAAH) mediate the antitumor activity of cannabidiol, a non-psychoactive cannabinoid.

It has been recently reported that cannabidiol (CBD), a non-psychoactive cannabinoid, is able to kill glioma cells, both in vivo and in vitro, independently of cannabinoid receptor stimulation. However, the underlying biochemical mechanisms were not clarified. In the present study, we performed biochemical analysis of the effect of CBD both in vivo, by using glioma tumor tissues excised from nude mice, and in vitro, by using U87 glioma cells. In vivo exposure of tumor tissues to CBD significantly decreased the activity and content of 5-lipoxygenase (LOX, by approximately 40%), and of its end product leukotriene B4 ( approximately 25%). In contrast cyclooxygenase (COX)-2 activity and content, and the amount of its end product prostaglandin E2, were not affected by CBD. In addition, in vivo treatment with CBD markedly stimulated ( approximately 175%) the activity of fatty acid amide hydrolase (FAAH), the main anandamide-degrading enzyme, while decreasing anandamide content ( approximately 30%) and binding to CB1 cannabinoid receptors ( approximately 25%). In vitro pre-treatment of U87 glioma cells with MK-886, a specific 5-LOX inhibitor, significantly enhanced the antimitotic effect of CBD, whereas the pre-treatment with indomethacin (pan-COX inhibitor) or celecoxib (COX-2 inhibitor), did not alter CBD effect. The study of the endocannabinoid system revealed that CBD was able to induce a concentration-dependent increase of FAAH activity in U87 cells. Moreover, a significantly reduced growth rate was observed in FAAH-over-expressing U87 cells, compared to wild-type controls. In conclusion, the present investigation indicates that CBD exerts its antitumoral effects through modulation of the LOX pathway and of the endocannabinoid system, suggesting a possible interaction of these routes in the control of tumor growth.

Anti-infective treatment of bacterial urinary tract infections.

Bacterial urinary tract infections (UTI) are frequently found in the outpatient as well as in the nosocomial setting. The bacterial UTI can be stratified into uncomplicated and complicated UTI. Antibiotic resistance is continuously increasing in uncomplicated as well as complicated UTI. In uncomplicated UTI efforts are made to use antibiotic substances exclusively for this indication. In complicated UTI as broad spectrum antibiotics are increasingly used, the higher the antimicrobial resistance rates are reported. There are two predominant aims in the antimicrobial treatment of both uncomplicated and complicated UTI: 1.) rapid and effective response to therapy, prevention of complications and prevention of recurrence in the individual patient treated, and 2.) prevention of emergence of resistance to anti-infective agents in the microbial environment. The use of antibiotics has to keep up with the continuous change in antimicrobial resistance and the tailored needs in the individual patient. Antibiotic substances therefore need to become evaluated for each indication and continuously followed for clinical usage. The knowledge of structure-activity relationships of antimicrobial substances and bacterial resistance mechanisms to antibiotics help to use antibiotics better in daily routine and design new derivatives and substances. The aim of this review is to describe the chemistry and structure-activity relationships of current antibiotics and promising substances in development for the treatment of UTI.

Growth factors and chemokines: a comparative functional approach between invertebrates and vertebrates.

Growth factors and cytokines control and coordinate a broad spectrum of fundamental cellular functions, and are evolutionarily conserved both in vertebrates and invertebrates. In this review, we focus our attention on the functional phylogenetic aspects of growth factors/cytokines like the Transforming Growth Factor-beta (TGF-beta), the Connective Tissue Growth Factor (CTGF), and the Vascular Endothelial Growth Factor (VEGF). We will also delve into the activites of two chemokine families, interleukin (IL)-8 (or CXCL8) and CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2). These molecules have been selected for their involvement in immune responses and wound healing processes, where they mediate and finely regulate various regeneration processes like angiogenesis or fibroplasia, not only in vertebrates, but also in invertebrates.

Distinctive cellular roles for novel protein kinase C isoenzymes.

A number of in vitro studies have implicated protein kinase Cdelta (PKCdelta) and PKCepsilon in the regulation of the immune system. In recent years, this has been convincingly demonstrated in mice deficient for PKCdelta and PKCepsilon. The reported phenotypes for these transgenic mice indicate that PKCdelta suppresses immunoresponsiveness and inhibits the proliferation of B-lymphocytes, while PKCepsilon is required for macrophages to mount an effective immune response to bacterial pathogens. In either case, these isoenzymes appear to cooperate in fine-tuning certain immunoreactions by either suppressing (PKCdelta) or stimulating (PKCepsilon) the transcription of various cytokines. This review will compare and contrast the structures of these two nPKC isoenzymes and their respective roles in the modulation of cytokine production and various other cellular processes, such as growth, differentiation, apoptosis, and tumor suppression.

Hematopoietic cell formation in leech wound healing.

The angiogenic process in vertebrates and hirudineans has been compared. The leech Hirudo medicinalis, subjected to an angiogenic stimulus (surgical explant or cytokine treatment) responds, as a vertebrate, with the formation of an extensive network of new vessels accompanied by the production of circulating cells. The reviewed data confirm the surprising similarity between hirudinean and vertebrate processes in wound healing, and suggest that basic common events such as antigenic expressions of endothelial and hemopoietic cells, cytokine secretion and regulation as well as extracellular matrix interactions, are conserved and extended across diverse species, tissues and developmental phases.

Structure and function of the extraembryonic membrane persisting around the larvae of the parasitoid Toxoneuron nigriceps.

The embryo of Toxoneuron nigriceps (Hymenoptera, Braconidae) is surrounded by an extraembryonic membrane, which, at hatching, releases teratocytes and gives rise to a cell layer embedding the body of the 1st instar larva. This cell layer was studied at different developmental times, from soon after hatching up to the first larval moult, in order to elucidate its ultrastructural, immunocytochemical and physiological function. The persisting "larval serosa" shows a striking structural and functional complexity: it is a multifunctional barrier with protective properties, limits the passage of macromolecules and it is actively involved in the enzymatic processing and uptake of nutrients. The reported results emphasizes the important role that the embryo-derived host regulation factors may have in parasitism success in Hymenoptera koinobionts.

Antitumor activity of endostatin against carcinogen-induced rat primary mammary tumors.

Endostatin, a Mr 20,000 fragment of collagen XVIII, potently inhibits the growth of experimental tumors implanted in mice. Here we report the cloning, expression, and antitumor activity of the rat form of endostatin. When tested on breast carcinomas arising in female virgin rats after intragastric administration of 9,10-dimethyl-1,2-benzanthracene (DMBA), endostatin induced significant inhibition of mammary tumor growth in all of the treated rats during a 4-week treatment period without signs of systemic toxicity. Interestingly, this arrest of tumor growth persisted throughout a four-week off-therapy period. Moreover, endostatin was effective in counteracting the development of multiple primary tumors. These results confirm that rat endostatin is a potent anticancer agent in a carcinogen-induced, spontaneously arising rat breast cancer model. It not only stops the growth of existing tumors but also decreases the incidence of the development of multiple neoplastic lesions.

PKCdelta acts as a growth and tumor suppressor in rat colonic epithelial cells.

We have analysed the expression of three calcium-independent isoforms of protein kinase C (PKC), PKCdelta, PKCepsilon and PKCzeta, in an in vitro model of colon carcinogenesis consisting of the nontumorigenic rat colonic epithelial cell line D/WT, and a derivative src-transformed line D/src. While PKCzeta and PKCepsilon showed similar protein levels, PKCdelta was markedly decreased in D/src cells when compared to the D/WT line. To assess whether down-regulation of PKCdelta was causally involved in the neoplastic phenotype in D/src cells, we prepared a kinase-defective mutant of PKCdelta. Stable transfection of this sequence caused morphological and growth changes characteristic of partial transformation in D/WT cells. Moreover, to test whether PKCdelta was involved in growth control and transformation in this model, we overexpressed PKCdelta in D/src cells. Transfected cells underwent marked growth and morphological modifications toward the D/WT phenotype. In a late stage in culture, transfected cells ceased to proliferate, rounded up and degenerated into multinucleated, giant-like cells. We conclude that PKCdelta can reverse the transformed phenotype and act as a suppressor of cell growth in D/src cells. Moreover, our data show that downregulation of this isoenzyme of PKC may cooperate in the neoplastic transformation induced by the src oncogene in D/WT cells.

Overexpression of protein kinase C epsilon is oncogenic in rat colonic epithelial cells.

We have analysed the expression of five protein kinase C [PKC] isoforms in an in vitro model using nontumorigenic rat colonic epithelial cells FRC/TEX CL D [D/WT] and in the related tumorigenic Ha-ras-transformed FRC/TEX CL D/H-ras line [D/ras]. The PKC subspecies alpha, delta, epsilon and xi were expressed at the protein level in both D/WT and D/ras cells, while beta PKC was undetectable in both lines. The levels of expression of the delta and xi isoforms were similar in D/WT and D/ras cells. Alpha PKC expression was decreased and epsilon PKC was increased in D/ras cells compared to the D/WT line. To assess whether overexpression of epsilon PKC was linked to the transformed phenotype, we have generated from D/WT cells two clones (D/epsilon-5 and D/epsilon-9) which stably overexpress epsilon PKC about fivefold. Overexpression of epsilon PKC caused marked morphological changes in both transfected clones, which were accompanied by increased saturation densities and anchorage-independent colony formation in semisolid agar. These growth effects were attenuated or reversed by chronic incubation with phorbol 12-myristate 13-acetate. Furthermore, D/epsilon-5 and D/epsilon-9 cells formed tumors in athymic nude mice with 100% incidence while the parental D/WT or vector alone (D/MV12) controls produced no tumors. We conclude that epsilon PKC can act as an oncoprotein when modestly overproduced in nontumorigenic D/WT colonic cells, and that this isoform of PKC may be linked to ras-modulated signal transduction leading to neoplastic transformation in colonic epithelium.

Protein kinase Cepsilon is oncogenic in colon epithelial cells by interaction with the ras signal transduction pathway.

We have shown previously that overexpression of the epsilon isoform of protein kinase C (PKCepsilon) in rat colonic epithelial cells causes malignant transformation, possibly by interacting with the ras signal transduction pathway (Oncogene 12: 847, 1996). We have now performed experiments to examine certain early steps in the ras signaling pathway. A marked increase of Raf-1 phosphorylation was detected in tumorigenic ras-transformed D/ras as well as in D/epsilon cells (overexpressing PKCepsilon), compared to the nontumorigenic D/WT parental line. Moreover, in the PKCepsilon-transformed D/epsilon cell line, stable transfection with a dominant-negative raf-1 (DNraf) sequence caused complete regression of the neoplastic phenotype. These results suggested that PKCepsilon-induced transformation was associated with increased Raf-1 activation, and that DNraf could block the oncogenic effect of PKCepsilon. Furthermore, transfection of D/WT cells with dominant-negative ras induced arrest of cell growth, and subsequent transfection with PKCepsilon cDNA enhanced cell proliferation and induced neoplastic transformation. These results suggest that ras acts upstream of PKCepsilon, and that overexpression of PKCepsilon circumvents the block in cell proliferation caused by dominant-negative ras. We conclude that PKCepsilon exerts its oncogenic activity in rat colonic cells by affecting the ras signaling cascade at the level of Raf-1 activation.

PKCs.

Molecular cloning and biochemical characterization of individual members of the protein kinase C (PKC) family demonstrated that single PKC isoenzymes display distinct structure, activation conditions, substrate specificity and tissue distribution. In the last few years a number of studies based on isoenzyme overexpression indicated that the PKC family coordinates a complex network of pivotal signal transduction pathways and pathway crosstalks, which regulate cell growth and differentiation, neoplastic transformation and malignant progression. This study reviews recent progress in PKC investigation, and focuses on achievements which have been shown to be relevant to cancer research.

Resistance of human leukemic cell lines to 1-beta-D-arabinofuranosylcytosine: characterization of an experimental model.

1-beta-D-arabinofuranosylcytosine (ara-C) is an antimetabolite used for the treatment of acute myelogenous leukemia. The ability of ara-C to kill neoplastic cells has been correlated to the induction of apoptosis. The clinical use of ara-C is limited by the development of drug resistance. Alterations in drug-induced apoptosis play a critical role in ara-C resistance. In particular, the proto-oncogene bcl-2 has been implicated in this phenomenon. To better understand the molecular basis of the role of bcl-2 in ara-C resistance, we investigated the relationship between the cytotoxic effect of ara-C, the expression levels and the subcellular localization of bcl-2 in three human leukemic cell lines (HL-60, KG1, J111). We have also evaluated the effects of ara-C on the J111 leukemic cell line (showing the lowest levels of Bcl-2 and the highest sensitivity to ara-C) overexpressing the bcl-2 oncogene. The model we developed here will allow further studies on the role of post-translational events involving bcl-2 (such as translocation and/or phosphorylation) in the cellular response to ara-C treatment.

Involvement of PI3K in PKCepsilon-mediated oncogenic signal in rat colonic epithelial cells.

We have recently demonstrated that overexpression of PKCepsilon is oncogenic in colonic epithelial cells. To test whether PI3K might be an upstream effector of PKCepsilon in cell transformation, we have overexpressed the p110alpha PI3K subunit in non-transformed D/WT colonic epithelial cells. Transfectants displayed the major in vitro features of transformed cells. Interestingly, no transformation occurred when p110alpha was co-transfected with a dead-kinase PKCepsilon mutant. The p85alpha subunit of PI3K, displaying a dominant-negative-like effect, was then transfected in PKCepsilon-transformed D/epsilon cells. The transformed profile of these cells was markedly reduced. To identify which by-products of PI3K might be involved in cell transformation we have transfected the D/WT cell line with cDNAs encoding the PI3 kinases hVps34 and C2beta. Overexpression of hVps34 did not cause cell transformation. Conversely, in vitro transformation was observed when C2beta was transfected into D/WT cells. These results indicate that phosphatidylinositol-3 monophosphate does not seem to be involved in cell transformation, and that phosphatidylinositol-3,4 bisphosphate and phosphatidylinositol-3,4,5 trisphosphate are more likely involved in this process. Thus, our data support the hypothesis of a linkage between PI3K and PKCepsilon, and indicate that PI3K may act as a source of second messengers responsible for oncogenic activation of PKCepsilon.

Differential phorbol ester growth modulation and protein-kinase-C isoenzyme expression in rat hepatoma-cells vs non neoplastic rat hepatocytes.

In the present study the expression of protein kinase C (PKC) isoenzymes and the growth modulation caused by phorbol esters were studied in two rat hepatic cell lines: the neoplastic MH1C1 hepatoma and the syngeneic immortalized non-neoplastic BRL3A cell line. Western blot analysis revealed that MH1C1 rat hepatoma cells express the a isoenzyme as the only isoform of PKC, whereas BRL3A cells showed immunoreactivity for alpha, delta and epsilon PKC. Immunoblot analysis showed that in both cell lines phorbol 12-myristate 13-acetate (PMA) caused a persistent, marked down-regulation of all expressed PKC isoenzymes. In MH1C1 cells, PMA also induced a reversible, marked dose-dependent growth inhibition, whereas a minimal impairment of cell proliferation was observed in BRL cells. The observed selective mitoinhibitory effect of PMA on tumor cells indicates PKC as an interesting target for innovative antiproliferative strategies based on the modulation of specific isoenzymes.

Analysis of calcium-dependent protein kinase-C isoenzymes in intrinsically resistant cloned lines of LoVo cells: reversal of resistance by kinase inhibitor 1-(5-isoquinolinylsulfonyl) 2-methylpiperazine.

In order to investigate the involvement of Protein Kinase C (PKC) in the signal transduction mechanisms related to intrinsic chemoresistance, two cellular clones were isolated from LoVo/WT colon adenocarcinoma cell line and their cytogenetic pattern was studied: LoVo C1.7 was intrinsically resistant to Doxorubicin while LoVo C1.5 showed the same resistance index as the mixed parental cell population. Two PKC isoforms, immunologically identified as beta and alpha PKC, were isolated from the cytosolic fraction of all cell types and one single peak of alpha PKC was obtained from the particulate fraction. Resistant LoVo C1.7 cells showed a significant increase of PKC activity; preincubation with H-7 induced PKC inhibition and reversal of drug resistance. These data suggest that in our cell system the identified calcium-dependent PKC subtypes can play a role in the mechanisms of intrinsic resistance.