MAPK11 (p38ß) is a major determinant of cellular radiosensitivity by controlling ionizing radiation-associated senescence: An in vitro study.

Background and purpose: MAPKs are among the most relevant signalling pathways involved in coordinating cell responses to different stimuli. This group includes p38MAPKs, constituted by 4 different proteins with a high sequence homology: MAPK14 (p38α), MAPK11 (p38ß), MAPK12 (p38γ) and MAPK13 (p38δ). Despite their high similarity, each member shows unique expression patterns and even exclusive functions. Thus, analysing protein-specific functions of MAPK members is necessary to unequivocally uncover the roles of this signalling pathway. Here, we investigate the possible role of MAPK11 in the cell response to ionizing radiation (IR). Materials and methods: We developed MAPK11/14 knockdown through shRNA and CRISPR interference gene perturbation approaches and analysed the downstream effects on cell responses to ionizing radiation in A549, HCT-116 and MCF-7 cancer cell lines. Specifically, we assessed IR toxicity by clonogenic assays; DNA damage response activity by immunocytochemistry; apoptosis and cell cycle by flow cytometry (Annexin V and propidium iodide, respectively); DNA repair by comet assay; and senescence induction by both X-Gal staining and gene expression of senescence-associated genes by RT-qPCR. Results: Our findings demonstrate a critical role of MAPK11 in the cellular response to IR by controlling the associated senescent phenotype, and without observable effects on DNA damage response, apoptosis, cell cycle or DNA damage repair. Conclusion: Our results highlight MAPK11 as a novel mediator of the cellular response to ionizing radiation through the control exerted onto IR-associated senescence.

p38ß (MAPK11) mediates gemcitabine-associated radiosensitivity in sarcoma experimental models.

BACKGROUND AND PURPOSE: Gemcitabine is an antitumour agent currently used in the treatment of several types of cancer with known properties as a radiosensitizer. p38MAPK signalling pathway has been shown to be a major determinant in the cellular response to gemcitabine in different experimental models. However, the molecular mechanism implicated in gemcitabine-associated radiosensitivity remains unknown. MATERIALS AND METHODS: The human sarcoma cell lines A673 and HT1080, and a mouse cell line derived from a 3-methylcholanthrene induced sarcoma were used as experimental models. Modulation of p38MAPKs was performed by pharmacological approaches (SB203580) and genetic interference using lentiviral vectors coding for specific shRNAs. Viability was assessed by MTT. Gene expression was evaluated by western blot and RT-qPCR. Induction of apoptosis was monitored by caspase 3/7 activity. Response to ionizing radiation was evaluated by clonogenic assays. RESULTS: Our data demonstrate that chemical inhibition of p38MAPK signalling pathway blocks gemcitabine radiosensitizing potential. Genetic interference of MAPK14 (p38α), the most abundantly expressed and best characterized p38MAPK, despite promoting resistance to gemcitabine, it does not affect its radiosensitizing potential. Interestingly, specific knockdown of MAPK11 (p38ß) induces a total loss of the radiosensitivity associated to gemcitabine, as well as a marked increase in the resistance to the drug. CONCLUSION: The present work identifies p38ß as a major determinant of the radiosensitizing potential of gemcitabine without implication of p38α, suggesting that p38ß status should be analysed in those cases in which gemcitabine is combined with ionizing radiation.

P53 pathway is a major determinant in the radiosensitizing effect of Palbociclib: Implication in cancer therapy.

Targeting cell cycle has become one of the major challenges in cancer therapy, being Palbociclib, a CDK4/6 inhibitor, an excellent example. Recently, it has been reported that Palbociclib could be a novel radiosensitizer agent. In an attempt to clarify the molecular basis of this effect we have used cell lines from colorectal (HT29, HCT116) lung (A549, H1299) and breast cancer (MCF-7). Our results indicate that the presence of a p53 wild type is strictly required for Palbociclib to exert its radiosensitizing effect, independently of the inhibitory effect exerted on CDK4/6. In fact, abrogation of p53 in cells with functional p53 blocks the radiosensitizing effect of Palbociclib. Moreover, no radiosensitizing effect is observed in cells with non-functional p53, but restoration of p53 function promotes radiosensitivity associated to Palbociclib. Furthermore, the presence of Palbociclib blocks the transcriptional activity of p53 in an ATM-dependent-fashion after ionizing radiation exposure, as the blockage of p21/WAF1 expression demonstrates. These observations are a proof of concept for a more selective therapy, based on the combination of CDK4/6 inhibition and radiotherapy, which would only benefit to those patients with a functional p53 pathway.

Blockage of autophagic flux is associated with lymphocytosis and higher percentage of tumoral cells in chronic lymphocytic leukemia of B cells.

PURPOSE: Autophagy has lately emerged as an important biological process with implications in several hematological pathologies. Recently, a growing body of evidence supports a putative role of autophagy in chronic lymphocytic leukemia; however, no definitive clue has been established so far. To elucidate this issue, we have developed a pilot study to measure autophagic flux in peripheral blood mononuclear cells from chronic lymphocytic leukemia patients, and explored its correlation with classical clinical/analytical parameters. METHODS/PATIENTS: Thirty-three chronic lymphocytic leukemia patients participated in the study. Autophagic flux in peripheral blood mononuclear cells was determined by western blot measuring the levels of the proteins p62 and lipidated LC3. Moreover, p62 mRNA levels were analyzed by RT-qPCR. RESULTS: Lymphocytosis and the percentage of tumoral lymphocytes in chronic lymphocytic leukemia patients statistically correlate with a blocked autophagic flux. CONCLUSION: Alterations in autophagic flux could play an important role in the physiopathology of chronic lymphocytic leukemia.

CHK1 expression in Gastric Cancer is modulated by p53 and RB1/E2F1: implications in chemo/radiotherapy response.

Radiation has a limited but relevant role in the adjuvant therapy of gastric cancer (GC) patients. Since Chk1 plays a critical function in cellular response to genotoxic agents, we aimed to analyze the role of Chk1 in GC as a biomarker for radiotherapy resistance. We analyzed Chk1 expression in AGS and MKN45 human GC cell lines by RT-QPCR and WB and in a small cohort of human patient's samples. We demonstrated that Chk1 overexpression specifically increases resistance to radiation in GC cells. Accordingly, abrogation of Chk1 activity with UCN-01 and its expression with shChk1 increased sensitivity to bleomycin and radiation. Furthermore, when we assessed Chk1 expression in human samples, we found a correlation between nuclear Chk1 accumulation and a decrease in progression free survival. Moreover, using a luciferase assay we found that Chk1's expression is controlled by p53 and RB/E2F1 at the transcriptional level. Additionally, we present preliminary data suggesting a posttranscriptional regulation mechanism, involving miR-195 and miR-503, which are inversely correlated with expression of Chk1 in radioresistant cells. In conclusion, Chk1/microRNA axis is involved in resistance to radiation in GC, and suggests Chk1 as a potential tool for optimal stratification of patients susceptible to receive adjuvant radiotherapy after surgery.

Papel de las proteínas quinasas activadas por mitógenos (MAPK) en el carcinoma de células renales esporádico / Role of Mitogen-Activated Protein Kinase (MAPK) in the Sporadic Renal Cell Carcinoma

Contexto: Últimamente, basándose en la implicación del gen supresor vhl en los casos de carcinoma de células renales (CCR), se ha evaluado la implicación de la ruta de señalización entre pVHL y el factor inducible por hipoxia 1 alfa (HIF-1α), ante la necesidad de encontrar nuevos marcadores diagnósticos, pronósticos y de respuesta a fármacos. Síntesis de evidencia: La sobreexpresión de HIF-1α confiere mejor pronóstico en pacientes afectos de CCR de tipo células claras (ccRCC). Además HIF-1α regula otros genes, concretamente el de la anhidrasa carbónica IX (CA-IX), cuya sobreexpresión es prácticamente exclusiva de los ccRCC y su determinación útil para el diagnóstico de este subtipo. Sin embargo, no se ha demostrado la implicación de CA-IX ni en el pronóstico ni en la respuesta a inmunomoduladores o antiangiogénicos. Ello hace necesario la evaluación global de toda esta ruta: pVHL → HIF-1α → CA-IX, e incluso el análisis de otras proteínas y vías de señalización que también controlan la actividad de HIF-1α. En este último caso, las MAPK, son críticas en la activación de HIF-1α, existiendo evidencias a nivel experimental del control sobre su actividad, aunque no se ha establecido su papel clínico como biomarcador. Si bien está demostrado el papel de las MAPK en los fenómenos de resistencia a quimio y radioterapia convencional, no lo está en la respuesta a sorafenib, dato llamativo si tenemos en cuenta que es inhibidor de varias proteín quinasas. Recientemente se ha observado que las MAPK pueden estar implicadas en la respuesta a distintas terapias, incluidas las basadas en inhibidores de tirosín quinasa. Conclusiones: La confirmación de estos datos supondrá una explicación a la variación observada entre pacientes, que con una misma alteración funcional del gen vhl presentan un distinto comportamiento biológico y clínico, y a una mejor selección de terapias no quirúrgicas (AU)

Context: Only on the basis of the involvement of the vhl suppressor gene in the cases of renal cell carcinomas (RCC), the involvement of the signaling pathway between the pVHL and the Hypoxia inducible factor 1, alpha (HIF-1α) has been evaluated because of the need to find new diagnostic and prognostic and response to drugs markers. Evidence synthesis: The overexpression of HIF-1α confers better prognosis in clear cell type RCC (ccRCC). Furthermore, HIF-1α regulates other genes, specifically that of the carbon anhydrase IX (CA-IX), whose overexpression is practically only of the ccRCC and its determination is useful for this subtype. However, the involvement of the CA-IX has not been demonstrated in the prognosis or in the response to immunomodulators or antiangiogenics. Therefore, it is necessary to make a global evaluation of all this pathway: pVHL → HIF-1α → CA-IX, and even the analysis of other proteins and signaling pathways that also control the HIF-1α activity. In the latter case, the MAPK are critical in the HIF-1α activation, there being evidence on the experimental level of the control on its activity. although its clinical role as a biomarkers has not been established. Although the role of the MAPK in the phenomena of resistance to conventional chemotherapy and radiotherapy has been demonstrated, it has not been demonstrated in response to sorafenib, an important piece of information if we consider that it is an inhibitor of several protein kinases. Recently, it has been observed that the MAPK may be involved in the responses to different therapies, included those based on tyrosine kinase inhibitors. Conclusions: The confirmation of these data would suppose an explanation of the variation observed between patients who, with the same functional alteration of the vhl gene, have a different biological, clinical behavior and better selection of non-surgical therapies (AU)

P38MAPK is a major determinant of the balance between apoptosis and autophagy triggered by 5-fluorouracil: implication in resistance.

5-Fluorouracil (5-FU), together with other drugs such as oxaliplatin, is one of the most important pharmacological agents in the treatment of colorectal cancer. Although mitogen-activated protein kinases (MAPKs) have been extensively connected with resistance to platinum compounds, no role has been established in 5-FU resistance. Here we demonstrate that p38MAPK activation is a key determinant in the cellular response to 5-FU. Thus, inhibition of p38MAPKα by SB203580 compound or by short-hairpin RNA interference-specific knockdown correlates with a decrease in the 5-FU-associated apoptosis and chemical resistance in both HaCaT and HCT116 cells. Activation of p38MAPK by 5-FU was dependent on canonical MAP2K, MAPK kinase (MKK)-3 and MKK6. In addition, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) showed a redundancy of function for the final activation of p38MAPK. Resistance associated with p38MAPK inhibition correlates with an autophagic response that was mediated by a decrease in p53-driven apoptosis, without effect onto p53-dependent autophagy. Moreover, the results with colorectal cancer-derived cell lines with different p53 status and patterns of resistance to 5-FU suggest that de novo and acquired resistance was controlled by similar mechanisms. In summary, our data demonstrate a critical role for the p38MAPK signaling pathway in the cellular response to 5-FU by controlling the balance between apoptosis and autophagy.

[Role of mitogen-activated protein kinase (MAPK) in the sporadic renal cell carcinoma]. / Papel de las proteínas quinasas activadas por mitógenos (MAPK) en el carcinoma de células renales esporádico.

CONTEXT: Only on the basis of the involvement of the vhl suppressor gene in the cases of renal cell carcinomas (RCC), the involvement of the signaling pathway between the pVHL and the Hypoxia inducible factor 1, alpha (HIF-1α) has been evaluated because of the need to find new diagnostic and prognostic and response to drugs markers. EVIDENCE SYNTHESIS: The overexpression of HIF-1α confers better prognosis in clear cell type RCC (ccRCC). Furthermore, HIF-1α regulates other genes, specifically that of the carbon anhydrase IX (CA-IX), whose overexpression is practically only of the ccRCC and its determination is useful for this subtype. However, the involvement of the CA-IX has not been demonstrated in the prognosis or in the response to immunomodulators or antiangiogenics. Therefore, it is necessary to make a global evaluation of all this pathway: pVHL → HIF-1α → CA-IX, and even the analysis of other proteins and signaling pathways that also control the HIF-1α activity. In the latter case, the MAPK are critical in the HIF-1α activation, there being evidence on the experimental level of the control on its activity. although its clinical role as a biomarkers has not been established. Although the role of the MAPK in the phenomena of resistance to conventional chemotherapy and radiotherapy has been demonstrated, it has not been demonstrated in response to sorafenib, an important piece of information if we consider that it is an inhibitor of several protein kinases. Recently, it has been observed that the MAPK may be involved in the responses to different therapies, included those based on tyrosine kinase inhibitors. CONCLUSIONS: The confirmation of these data would suppose an explanation of the variation observed between patients who, with the same functional alteration of the vhl gene, have a different biological, clinical behavior and better selection of non-surgical therapies.

High prevalence of human papillomavirus 16 in penile carcinoma.

In order to analyze the incidence and prevalence of Human Papillomavirus (HPV) in penile carcinoma, we studied 49 patients with penile carcinoma. Formalin-fixed, paraffin-embedded tissue samples were collected from 64 samples of penile carcinoma from the Hospital General Universitario (Albacete, Spain). Cases were histologically classified and the polymerase chain reaction (PCR) method was used to detect the presence of HPV. Two sets of consensus primers were used, the My09/My11, and the GP5+/GP6+. All positive cases were sequenced in order to establish the implicated genotype. Our results showed that 38 of the 49 cases were positive for HPV (77,5%). HPV16 appeared in 32 (84,2 %) of the 38 positive cases and HPV18 in 4 (10,5%). Our data demonstrate that the My09/My11 primers are more sensitive than GP5+/GP6+ primers, although the combination of the two sets of primers notably increased the total number of HPV positive cases detected.

Adenovirus E1A orchestrates the urokinase-plasminogen activator system and upregulates PAI-2 expression, supporting a tumor suppressor effect.

Invasiveness and metastatic potential are the two most important properties defining malignancy. The adeno-virus E1A (Ad-E1A) gene has a dual effect as a proliferative gene and as a tumor-suppressor gene, decreasing tumor growth and the metastatic potential of malignant cells. In order to study genes related with the antimetastatic effect of Ad-E1A in human cells, we performed a microarray analysis using OncoChiptrade mark. In three independent experiments, NIH3T3, IMR90 and MDA MB 435 cells were infected with pLPC retroviruses carrying the adenovirus 12S E1A gene or the GFP gene. We analyzed cDNA expression by using the CNIO OncoChipTM, a cDNA microarray containing a total of 6386 genes represented by 7237 clones. uPA, uPAr, tPA, PAI-1 and PAI-2 were also studied at RNA and protein levels. Microarrays of cDNA expression, RT-PCR and Western blot performed in IMR90 E1A-expressing cells showed downregulation of uPA, uPAr, tPA, PAI-1 and upregulation of PAI-2. These results were confirmed in NIH3T3 and MDA MB 435 breast carcinoma cells, with PAI-2 upregulation by RT-PCR and Western blot. In addition, zymographic analysis demonstrated that E1A expression greatly reduced the gelatinase activity of the pro-MMP2 and -MMP9 proteins. We propose that adenovirus E1A may orchestrate the expression of most members of the urokinase-plasminogen activation system, downregulating potentially invasive genes and upregulating PAI-2, which is associated with a better prognosis in human tumors.

PKB/Akt media la radio sensibilidad asociada a Ataxia Telangiectasia / PKB/Akt mediates radiosensitivity associated to ataxia telangiectasia

El gen mutado en el síndrome de Ataxia Telangiectasia está implicado en múltiples funciones celulares queabarcan desde la respuesta al daño genotóxico causado por la radiación ionizante hasta la respuesta a insulina.Curiosamente la ruta de señalización mediada por Akt esta relacionada con los mismos estímulos. En el presenteestudio demostramos como el gen mutado en Ataxia Telangiectasia (ATM) controla la fosforilación deAkt, en respuesta a estímulos como la radiación ionizante o la insulina. Estos datos han sido obtenidos usandomodelos experimentales como células derivadas de enfermos de Ataxia o de ratones nulos para la expresión deATM. Nuestro estudio propone nuevas explicaciones para entender la radio sensibilidad característica de estosenfermos y destaca el papel primordial de Akt en la respuesta a la radiación ionizante

The gene mutated in ataxia telangiectasia (ATM) has been implicated in several functions such as cell cycle, response to DNA damage, and insulin. Curiously, the PKB/Akt-mediated signaling route is related to the same cellular responses. We show in this work that ATM is a major determinant of full PKB/Akt activation in response to insulin or gamma-radiation. This conclusion was inferred from the results obtained in transient transfection assays using exogenous PKB/Akt and ATM in Cos cells, and also in cell lines derived from ataxia telangiectasia patients or KO mice. Our study proposes new clues to understand the radiosensitivity associated to ataxia telangiectasia and supports a critical role for PKB/Akt in the cellular response to ionizing radiation

The calcium-binding protein calretinin is a marker of the companion cell layer of the human hair follicle.

BACKGROUND: Ultrastructural studies of the hair follicle show that the outer root sheath (ORS) does not consist of a homogeneous cell population. The innermost cell layer of the ORS, also called the companion layer, is a single cell layer closely associated with the Henle layer of the inner root sheath. OBJECTIVES: To describe the immunohistochemical expression of calretinin, a calcium-binding protein, in the human hair follicle. METHODS: Immunohistochemical studies using two different antisera to calretinin were performed in paraffin-embedded and in frozen scalp specimens using standard techniques. RESULTS: Calretinin immunostaining was consistently and specifically found in the companion cell layer of hair follicles. CONCLUSIONS: These findings provide further evidence to support the notion that the companion layer is not only morphologically, but also immunohistochemically, different from the other cells of the ORS.

Selective effects of E1B-defective adenoviruses and adenovirus E1A mutants in deficient mouse primary embryonic fibroblasts.

E1B-defective adenoviruses have been described as exerting selective cytopathic effects on transformed cells. Previously, we showed that adenovirus dl118, lacking both E1B proteins, very efficiently kills most human malignant cell lines. In order to study whether these selective effects were due to selective replication of dl118 in cells harboring specific genetic alterations, we compared the viability of various deficient mouse primary fibroblasts. We studied mouse embryonic fibroblasts (MEFs) derived from p16, p21, p27 and p53 knockout mice, as well as wild-type MEFs. We infected them with 100 p.f.u. of adenoviruses adl118, adwt300, and adenoviruses carrying the E1A mutant 922 (the E1a product only binds to the p300 and related proteins) and Ad646 (the E1A product binds to the pRb and related proteins). The percentage of infectivity was evaluated with an adenovirus carrying the green fluorescent protein (AdGFP). With AdGFP, clear green fluorescent signals were detected in more than 70% of the cells after 3 days of infection. After infection with several adenoviruses, we observed that E1A mutant 922 killed all the MEFs. Conversely, the E1a mutant Ad646 exerted its major effects on control wild-type MEFs. Moreover, Adl118 killed the wtMEFs and other MEFs slightly more efficiently than did wtAd, but less than Ad922. No viral replication was detected by adding the obtained supernatants to HEK293 cells. Due to the absence of significant viral replication on these cells, the results could be interpreted as direct effects of E1A and E1A mutant proteins on the different mouse cells carrying diverse genetic alterations.

Intravascular injections of a conditional replicative adenovirus (adl118) prevent metastatic disease in human breast carcinoma xenografts.

We describe a study showing that the adenovirus adl118, lacking both E1B proteins, very efficiently kills human malignant cells 'in vitro' and 'in vivo'. Since many breast cancer patients do not have metastasis at the time of diagnosis, but finally develop it, we planned to study whether intravascular injections of adl118 could prevent metastatic development. We studied the effects of this mutant adenovirus in an orthotopic model of human breast carcinoma xenografts with the breast MB435-lung 2 cell line, which is highly metastatic in the lungs. In this study, all primary tumors were excised when they reached 50-100 mm3 volume in the animals. After surgery, 10(10) p.f.u. of adl118 was intravenously injected into a random group of animals, either three times during the first week only, or once every week. At death, almost all the control animals showed numerous lung metastases of large size, which were present in only 15-40% of the treated animals, depending on the size of the primary tumor at the time of excision. Overall survival was 50-70 days in control mice, and over 120 days in mice injected with adl118. Concomitant treatment with adl118 and cisplatin did not enhance the antitumor effects of adl118. With these results, we conclude that intravenous injection of conditional replicative adenovirus, after excision of the primary tumor, induces a clear decrease in the metastatic disease, and could be a new strategy in preventing tumor metastasis of breast carcinomas.

Tumor heterogeneity: morphological, molecular and clinical implications.

Malignant tumors are characterized by their great heterogeneity and variability. There are hundreds of different types of malignant tumors that harbour many oncogenic alterations. The tumor heterogeneity has important morphological, molecular and clinical implications. Except for some hematopoietic and lymphoproliferative processes and small cell infant tumors, there are not specific molecular alterations for most human tumors. In this review we summarize the most important aspects of carcinogenesis and chemoradiosensitivity of malignant cells. In this regard, some oncogenes such as neu, ras and bcl-2 have been associated with cellular resistance to treatment with anticancer agents. The knowledge of oncogenic alterations involved in each tumor can be important to correlate the morphological features, the genetic background, the prognosis and the clinical response to treatment with anticancer agents. Based on the molecular background of the tumor there are new cancer gene therapy protocols. For example using adenovirus Ela in tumors with overexpression of neu oncogene, inhibitors of tyrosine kinase specific for the PDGF receptor in glioma, inhibitors of farnesil transferase to prevent ras activity in tumors with mutations in the ras gene.

A role for the p38 mitogen-acitvated protein kinase pathway in the transcriptional activation of p53 on genotoxic stress by chemotherapeutic agents.

The tumor suppressor p53 plays a central role in sensing damaged DNA and orchestrating the consequent cellular responses. However, how DNA damage leads to the activation of p53 is still poorly understood. In this study, we have found that the p38 mitogen-activated protein kinase (MAPK) plays a key role in the activation of p53 by genotoxic stress when provoked by chemotherapeutic agents. Indeed, we found that blockade of p38 prevents stimulation of the transcriptional activity of p53 and that activation of the p38 pathway is sufficient to stimulate p53 function. Furthermore, we observed that p38 does not affect the accumulation of p53 in response to DNA damage or its nuclear localization. In contrast, we observed that p38 associates physically with p53, and we provide evidence that this MAPK phosphorylates the NH2-terminal transactivation domain of p53 in serine 33, thereby stimulating its functional activity. Moreover, inhibition of the p38 MAPK diminished the apoptotic fraction of cells exposed to chemotherapeutic agents and increased cell survival, thus suggesting a role for p38 activation in the apoptotic response to genotoxic stress when elicited by drugs used in cancer therapy.

Adenovirus lacking the 19-kDa and 55-kDa E1B genes exerts a marked cytotoxic effect in human malignant cells.

UNLABELLED: The adenovirus (Ad) E1A gene exerts an antitumor effect and can induce sensitivity to treatment with DNA-damaging agents. In contrast, the Ad 19-kDa E1B protein inhibits E1A-mediated apoptosis and the 55-kDa E1B inactivates the p53 protein. In this paper, we study the in vitro and in vivo effects of a 19-kDa and 55-kDa E1B-defective Ad in several malignant human tumor cell lines. MATERIALS AND METHODS: Nontumorigenic human fibroblasts (CCD-45SK and Hs67), peripheral blood lymphocytes, and several human tumor cell lines derived from cervix, colon, and breast carcinomas, epidermoid carcinoma, and osteosarcoma (HeLa, HT29, MCF7, Saos-2, and A431 cell lines) were studied. Wild-type (wt) Ad type 5 and H5 dL118 Ad, a mutant with the deleted E1B region, were employed. The cells were infected at 20 plaque-forming units, and cell viability was evaluated by the crystal violet method. In the in vivo experiments, 2 x 10(6) cells from the carcinoma cell lines HeLa, A431 and HT29 were injected into nude mice. The tumorigenicity of previously infected cells and after an intratumoral injection of Ad was analyzed. The mice received whole-body gamma-irradiation. RESULTS: The H5 dL118 mutant produced a marked cytopathic effect in all of the malignant cells, surpassing that of the wt Ad; viability at 72 hours ranged from 11% to 20% for H5 dL118 Ad and from 70% to 93% for the wt Ad with respect to uninfected controls. In the in vivo experiments, a total inhibition of tumorigenicity was detected when cells were infected prior to injection and a partial and transitory decrease in tumorigenicity was detected when the mutant H5 dL118 was injected intratumorally. gamma-irradiation enhanced the in vivo antitumor effects. CONCLUSIONS: These results indicate that infection with completely E1B-deficient Ads induced a marked cytopathic effect on malignant cells that was higher than that seen for wt Ads; in addition, infection with such Ads exerts a tumor suppressor effect in vivo.