PMC:5608921 / 14614-24632 JSONTXT

{"project":"2_test","denotations":[{"id":"28846079-24018418-79081919","span":{"begin":371,"end":373},"obj":"24018418"},{"id":"28846079-19209183-79081920","span":{"begin":375,"end":377},"obj":"19209183"},{"id":"28846079-8610145-79081921","span":{"begin":520,"end":522},"obj":"8610145"},{"id":"28846079-15699322-79081922","span":{"begin":524,"end":526},"obj":"15699322"},{"id":"28846079-21502497-79081923","span":{"begin":528,"end":530},"obj":"21502497"},{"id":"28846079-19741607-79081924","span":{"begin":658,"end":659},"obj":"19741607"},{"id":"28846079-20804975-79081925","span":{"begin":661,"end":662},"obj":"20804975"},{"id":"28846079-16597322-79081926","span":{"begin":879,"end":881},"obj":"16597322"},{"id":"28846079-16597322-79081927","span":{"begin":960,"end":962},"obj":"16597322"},{"id":"28846079-8610145-79081928","span":{"begin":1751,"end":1753},"obj":"8610145"},{"id":"28846079-15699322-79081929","span":{"begin":1755,"end":1757},"obj":"15699322"},{"id":"28846079-8390671-79081930","span":{"begin":2099,"end":2101},"obj":"8390671"},{"id":"28846079-11063126-79081931","span":{"begin":2241,"end":2243},"obj":"11063126"},{"id":"28846079-9790766-79081932","span":{"begin":2801,"end":2803},"obj":"9790766"},{"id":"28846079-10550545-79081933","span":{"begin":2805,"end":2807},"obj":"10550545"},{"id":"28846079-18548112-79081934","span":{"begin":3055,"end":3057},"obj":"18548112"},{"id":"28846079-7969162-79081935","span":{"begin":3397,"end":3399},"obj":"7969162"},{"id":"28846079-16282489-79081936","span":{"begin":3401,"end":3403},"obj":"16282489"},{"id":"28846079-24983759-79081937","span":{"begin":4181,"end":4183},"obj":"24983759"},{"id":"28846079-11390614-79081938","span":{"begin":4483,"end":4485},"obj":"11390614"},{"id":"28846079-11063126-79081939","span":{"begin":4790,"end":4792},"obj":"11063126"},{"id":"28846079-19439729-79081940","span":{"begin":5496,"end":5498},"obj":"19439729"},{"id":"28846079-8248223-79081941","span":{"begin":5500,"end":5502},"obj":"8248223"},{"id":"28846079-17342777-79081942","span":{"begin":5739,"end":5741},"obj":"17342777"},{"id":"28846079-24418098-79081943","span":{"begin":6237,"end":6239},"obj":"24418098"},{"id":"28846079-2276176-79081944","span":{"begin":6537,"end":6539},"obj":"2276176"},{"id":"28846079-19047174-79081945","span":{"begin":6541,"end":6543},"obj":"19047174"},{"id":"28846079-19008331-79081946","span":{"begin":7595,"end":7597},"obj":"19008331"},{"id":"28846079-16456137-79081947","span":{"begin":7644,"end":7646},"obj":"16456137"},{"id":"28846079-19047174-79081948","span":{"begin":7648,"end":7650},"obj":"19047174"},{"id":"28846079-15208259-79081949","span":{"begin":7675,"end":7677},"obj":"15208259"},{"id":"28846079-16456137-79081950","span":{"begin":7810,"end":7812},"obj":"16456137"},{"id":"28846079-19047174-79081951","span":{"begin":7814,"end":7816},"obj":"19047174"},{"id":"28846079-23065599-79081952","span":{"begin":7845,"end":7847},"obj":"23065599"},{"id":"28846079-24196716-79081953","span":{"begin":8314,"end":8316},"obj":"24196716"},{"id":"28846079-19571878-79081954","span":{"begin":8613,"end":8615},"obj":"19571878"},{"id":"28846079-21866153-79081955","span":{"begin":8617,"end":8619},"obj":"21866153"},{"id":"28846079-22143790-79081956","span":{"begin":8621,"end":8623},"obj":"22143790"},{"id":"28846079-12525422-79081957","span":{"begin":9411,"end":9413},"obj":"12525422"},{"id":"28846079-22870269-79081958","span":{"begin":9813,"end":9815},"obj":"22870269"}],"text":"Discussion\n\nContribution of suprabasal epithelial cells to carcinogenesis and regeneration\nIn stratified epithelia, the proliferative compartment is restricted to the basal layer. Here, cycling stem cells and progenitors live together and derive into the non-dividing terminally differentiated cells of suprabasal layers which mature up to producing the cornified tissue.13, 23 Commonly, hyperplasia and dysplasia in skin and cervix are characterized by a high number of proliferating cells located in suprabasal layers.19, 20, 24 The recurrent hypothesis to this phenotype is the invasion of basal proliferating cells into upper layers. However, recent data5, 6 open alternative hypotheses that need to be considered to trace the origin of cancer-initiating cells.\nIt has been proposed that a productive cycle of HPV in the cervix starts with the infection of basal layer cells.16 Upon differentiation, suprabasal cells start to express the early viral genes.16 Therefore, the primary cells for E6/E7 action are suprabasal cells which, in response, re-enter into the cell cycle. Tg(K6b-E6/E7) mice reproduced this pattern in the cervix, and accordingly, cell proliferation was promoted in suprabasal layers. This function is active in the growing phases of the estrous cycle when K6b was detected to be highly expressed. Since suprabasal E6/E7 expression did not affect the number of proliferating basal cells, few of these cells contributed to the suprabasal proliferation pattern observed. Therefore, suprabasal cells are relevant candidates to carry the role of cancer-initiating cells in the cervix infected with HPV. In contrast, although Tg(K14-E6/E7) mice can reproduce many aspects of cervical–uterine cancer when chronically treated with E2,19, 20 the compelled premise for the search of cancer-initiating cells in these mice is that they are stem/progenitor cells of the basal layer. Therefore, these mice unlikely model the initiation of cervical carcinogenesis in humans infected with HPV as, possibly, Tg(K6b-E6/E7) mice do.\nIt is interesting that, in contrast with Tg(K14-E6/E7) mice,25 the interfollicular epidermis of Tg(K6b-E6/E7) mice is not affected and skin alterations are restricted to the hair follicle renewal cycle.26 This is expected because K6b is expressed in the companion layer of the inner root sheath of hair follicles and regulated during the renewal cycle. In contrast, upon ear punch injury, a marked increase in K6b and K16 expression was observed that correlates with the increased oncogene expression that, in the ear regenerating epidermal area with well-organized basal layer correlated with the restricted presence of K6b in suprabasal layers. This is similar to the growing cervical epithelium (Figure 1c) and the stimulated growing epidermis without injury.27, 28 The disorganized epidermis commonly seen in the growing area of regenerating ears is possibly due to the high contribution of migratory cells from the wound borders and a delay in the reconstruction of the basal layer at early regeneration stages.21 In agreement with our observations, the epidermis of Tg(K10-E6/E7) mice, with expected constitutive limited oncogene expression in suprabasal layers, showed hyperplasia/hyperkeratosis and dividing cells in suprabasal layers under untreated conditions but, in contrast, a marked number of dividing cells were also increased in basal layers.29, 30 This latter effect might be a long-term consequence of the constant re-entering into the cell cycle of suprabasal cells (see below), since neither hyperplasia in the interfollicular epidermis under untreated conditions nor increased basal cell proliferation were observed in Tg(K6b-E6/E7) mice.\nAn interesting observation was the large area of coincidence of K5 and K6b expression and the location of most dividing cells in the growing epithelial tissue. In particular, we found that the zone of coincidence of K6b and K5 expression in the cervical epithelial tissue correlates with the area where dividing cells in suprabasal layers were present. Accordingly, extension of K5 expression to upper layers has also been observed in organotypic raft cultures transfected with HPV.31 Therefore, K6b+/K5+ cells in the cervix might identify those susceptible for oncogene-induced cell cycle re-entry. Although we detected the highest amount of E7 in upper suprabasal layers, it has been reported that high levels of p21/p27 make differentiated cells refractory to E7 mitogenic effects.32\nIn Tg(K6b-E6/E7) mice, longer duration of the growing phases is the likely cause of the thicker cervical epithelium during proestrus–estrus phase. This is similar to the effect of E6/E7 on hair follicles, which also show higher cycling frequency and a longer growing phase (anagen) than those of WT mice.26 We interpreted this phenomenon as oncogenes preventing from the entry of stem/progenitor cells of the bulge into the G0 cell cycle phase, expected for resting hair follicles (telogen). An alternative interpretation accordingto the present study is that E6/E7 drives the re-entry into the cell cycle of postmitotic K6b+ cells causing hair follicle growth from these cells instead of the regular stem cells that receive the resting signals.\n\nEstradiol as a growth-promoting factor\nEstradiol is an endocrine factor required for the initiation of growth of the cervical–uterine tract, such that lack of ERα in pituitary is sufficient to interrupt estrous cycling and the cervical tissue remains at diestrus.33, 34 However, in agreement with the participation of additional factors in estrous cycle regulation, continuous administration of E2 at the dose used here did not prevent from the action of factors ending the growth phase (e.g., progesterone12) and cycling continued. Proestrus initiation in Tg(K6b-E6/E7) mice was completely dependent on estradiol signaling and the end of the growth phase occurred as in WT mice. This is likely due to the transient E6/E7 expression during the estrous cycle. Exogenous E2 did not increase the frequency of estrous cycles but endogenous estradiol was required to exit from diestrus in both WT and Tg(K6b-E6/E7) mice. Therefore, the reversible interruption of carcinogenesis by raloxifene recently reported35 could be due to the induced estrous cycle pause at diestrus, stage at which E6/E7 are expressed at low levels and, likely, the HPV life-cycle is halted in human patients.\nAn additional possible role of estradiol in the regulation of estrous cycle is the growth promoting activity during proestrus.36, 37 Accordingly, estradiol extended the time WT cervical tissue was at the proestrus–estrus phase. In addition, we observed that after proestrus initiation, ER inhibition caused a marked reduction in basal proliferating cells of the cervix. This same effect was noted in basal and suprabasal cells of the cervix of Tg(K6b-E6/E7) mice, revealing the cooperative effect of oncogenes and estradiol. This cooperation was confirmed by the cancerous phenotype generated in Tg(K6b-E6/E7) mice treated with E2, showed even under continuous cycling. Interestingly, similar growth effects were noted in regenerating ears of both WT and Tg(K6b-E6/E7) mice, though only a mild effect was noted in regeneration efficiency. However, estradiol does not seem to be a limiting factor in this growth promoting activity in female mice, since increasing estradiol levels did not produce a marked effect on regeneration efficiency or the number of proliferating cells. ERα is the best candidate to carry out this activity since mediates estradiol mitogenic activities in vitro38 and is expressed in the lower layers of cervix14, 37 and epidermis epithelia.39 However, an indirect effect mediated by estradiol signaling in the cervical stroma has been suggested based in the expression of ERα14, 37 and its genetic inactivation40 in this tissue. A definitive conclusion in this regard awaits further investigation, since the ERα gene inactivation strategy used to support this possibility did not discard that the gene was also deleted in endocrine organs (e.g., pituitary), consequently blocking the estrous cycle and the carcinogenic process as referred above.\n\nMechanisms of regeneration in carcinogenesis\nPostmitotic cells can contribute to tissue repair by dedifferentiation into stem cells.41 Our data support that the increased growth induced by HPV oncogenes at the initial stages of carcinogenesis is mostly due to the cell cycle re-entry of suprabasal cells without requiring dedifferentiation into basal-like cells (Figure 7) similar to what has been observed in regenerating tissues.42, 43, 44 Nonetheless, the re-entry into the cell cycle of postmitotic cells could be essential for dedifferentiation at later stages of carcinogenesis. Actually, this phenomenon might explain the increase in basal dividing cells in the epidermis of Tg(K10-E6/E7) mice as referred above. We propose that the long-term cooperation between E6/E7 and estradiol in cervical–uterine cancer development could result from the cumulative increase in new proliferating cells contributing to epithelial growth and carcinogenesis (Figure 7). In this regard, it was interesting to find an increased number of koilocyte-like and bi-nucleated cells in the chronically growing cervical epithelium of E2-treated Tg(K6b-E6/E7) mice, as occurs at the early stages of carcinogenesis in the HPV-infected human tissue.22\nCancer and regeneration are closely related processes. The present work supports this relationship showing that two well-known promoters of cancer, estradiol and HPV oncogenes, also promote regeneration. Furthermore, it is interesting the parallel estradiol-E6/E7 cooperation for growth observed between cervix and skin. Accordingly, as in the cervix, ERα has been implicated in skin carcinogenesis.45 Since ear regeneration is a simple experimental model, the data presented encourage further studies in regenerating ears of Tg(K6b-E6/E7) mice to understand the early stages in cervical carcinogenesis.\n"}