The above mentioned perpendicularity change involved a 90˚ turn of the 4n nucleus before division in this same polarity changed condition, which led to withdrawal of the old cyto-skeleton with result of destruction of cell-to-cell adherence proteins (i.e., E-cadherin and β-catenin, cancer well known),giving movable freedom of the first cells. A further consequence was that these cells were born in a perpendicular orientation to the surrounding normal cells, and that they had to rebuild a new cytoskeleton, changing cell polarity and cell shape (wa). In the literature a perpendicular whole cell change is ascribed to gain of embryological “epithelial-mesenchymal transition” process (EMT or MET) [37] with outcome of destroyed inter-cellular adherence proteins for movability, but certainly not with link to a rebuilding of the cytoskeleton and cell polarity and cell shape changes. Cell polarity changes have been suggested to be the “gate” to tumorigenesis [38]. But back to EMT/MET, a report on ovarian cancer cells in metas-tasis showed note, 4n dividing cells in a perpendicular orientation relative to the cytoskeleton (“cytoskeleton skewed tetraploid”), which released migratory cells with colonizing ability in other body organs [39], which throws serious doubt on the EMT hypothesis. Remarkably, this same type of perpendicularity of nuclear divisions was described from the extent unicellular organism Aulachantha and a bacteria [40] [41] which also was a feature in genomic doubling recombination repair, and furthermore, showed segregation of whole genomes. Evidential material for mammalian genome conservation of these archaic (pre-mi- totic/meiotic) nuclear/division traits comes from nutrition deficitDrosophila cells, claiming amitosis in the absence of a normal spindle apparatus [42]. This occurrence and our own observations of amitotic abnormalities in fitness-gained first cell proliferative activity with multicellular growth, suggested that DNA damage exposed cells, responded withawakening of evolutionary conserved,archaic division traits, pre-mitosis/-meiosis evolutionary division trait. This happening would put amitotic division traits into human cells with mitosis, leading to questions of adaptation or apoptosis occurrences? It was concluded from the abnormal whole complement segregations without normal anaphase behavior that an adaptation had occurred, which was called amitotic-mitosis.

In oral aggressive cancer the cytoskeleton showed “defects” [43], and in colon crypt with APC mutated gene, the resultant proliferative growth was found in measured 90˚ turn relative to the basal cell membrane [44] [45] which proceeded to the cancerous cell phenotype from tetraploid aneuploidy. The foregoing pre- cancer phase also showed accumulation of 4n, a feature in two other pre-cancers, ulcerative-colitis and Barrett’s esophagus [46] [47]. In addition the latter report showed that cytometric isolated 4n cells became cell culture “enriched” via a 2n cycling phase (4n > 2n > 4n > 2n > -etc.,) expressed by one tall peak for 4n cells and a new smaller peak with measured 2n ploidy cells. This latter remarkable observation in the absence of 4n, aneuploid divisions, demonstrated inherited capability of the diploid cells to instigate 4n cells, genome reductive behavior. Recently in soft tissue sarcoma cell lines, it was shown that there were over-ex- pression of some mitotic genes (the CINSARC phenotype) by highly mobile and invasive 4n cells, which did not express “proliferative advantage”, compared to the diploid cells present [48]. The authors theorized that the cytoskeleton was miss-behaving from the mitotic proteins, and developed a screen for drugs with anti-mitotic and anti-cytoskeleton effects, and thereby discovered that inhibition of “several mitotic kinases”, dramatically, impaired the “invasive and migratory properties” of the 4n cells. These in vivo “special” 4n cell behaviors, were no different from the observed behaviors of the in vitro GR4n-SDS cells, suggested to be a traceable model system in tumorigenesis. Notably,the 4n in vivo cells demonstrated therapy vulnerability with cancerous effect. These developments appear tumorigenesis more likely than the tumor driver idea (below), with the above therapeutic promise likely enhanced from molecular landscapes of the special 4n and fitness gained 2n cells.

Left is evidential proof for the first cells’ expressed “proliferative advantage”, the hallmark for tumorigenesis initiation from normal cells (H/WREFF). This was earlier done from glutamine deficiency experiment from proliferative first-cells [65]. Such cells were seeded at 7 - 800,000 cells into each T-25 flasks, with similar seeding from normal optimum, proliferating normal cells. Hemocytometer readings of growths at 3 days, showed normal cell values 1.7 and 2.2 × 10⁶ and experimental values, 3.4 and 3.7 × 10⁶ cells in two different readings each, which is low, and likely only expresses as moderate proliferation difference from normal cells. These initial measurements showed 5 million more cells in fitness-gain cultures than in the control cultures with the notable, cancer-associated characteristic of being multilayered growth. To this important in vitro cancer-asso- ciated observation, there had earlier been an observation of special 4n cell derived 3-D tumor-like spheres with polygonal cell shape changes,floating free in the liquid growth medium,or being attached to the cultural surface [66]. These photographic illustrated spheres/balls of cells, revealed in 5 - 6 week old senescent cultures, can be likened to in vivo senescence, colon adenoma and breast DCIS (ductal carcinoma in situ) pre-cancers, having “risk” of oncogenic transformation. Furthermore, both in vitro and in vivo “oncogenic” transfor-mations were preceded by senescence-associated telomere DNA damage from natural, time-dependent attritions, abundantly earlier shown to cause genomic instability, notably, breakage-fusion-bridge (B-F-B) cycles, well known in cancers [67]. From this latter development a pre-dicttion is that exposure of multilayered first cell growth to a cell killing agent (DNA damage), the recovery growth would show cell shape change, different from that in the multi-layered growth (fibroblastic-like) and, the growth might be 3-D-like under proper cultural con-ditions (soft agar?). The virus work above,undeniably demonstrated that successive death signaled cells responded with regrowth,which histological and morphological was cancer-alike to diagnostic and prognostic criteria used in cancer-pa- thology today. This never before obtained in vitro happening, cries loudly for further investigations. Don’t let this in vitro landmark occur-rence from GR4n- SDS derived first cells,be pushed “under the rug”, cancer is a deadly, patient- horrible disease, and not an intriguing biological issue, as said above. And,also,don’t let big time profit [8] [9] [12] hinder you from the likely disclosure of therapy relevant,novel information from molecular sequencing data of special 4n and 2n fitness-gained cells.

In Figure 1 three different cell cycle circles from mitosis to mitosis are shown: (A)—normal cell cycling, (B)—stress associated cycling, and (C)—suggestions of endoreplication models [68] [69] [70]. Left-out is so-called endomitosis in which the replicated cells goes into mitosis, but performs only anaphase A not B, followed by re-enter into S-period, which has been adopted from megakaryocyte cycles [71]. This particular polyploidization mechanism have been discussed in two recent reports [72] [73]. Herein the slippage process in cause of tetraploid cells is uniformly considered, mainly because of evidential documented deteriorated mitotic program, Cyclin B and kinase Cdk-1 destruction [74]. As mentioned mitotic slippage process is also a consequence from normal telomere attrition at senescence, which was associated with appearance of 46, 4-chroma-tid chromosomes [21] [22] [23] [24] [25]. The shortened uncapped telomeres (DNA damaged signaling) much earlier were shown to be associated with breakage-fu- sion-bridge (B-F-B) cycle, giving rise to dicentric chromosomes, which is a cancer occurrence [75].

Back to the normally depicted cell cycle (Figure 1(A)). In this cycle from one mitosis to the next, checkpoint controls guide against abnormalities, which are situated at the borders of “phase” changes. For example, the checkpoints at

G2/M and at G1/S have received a lot of attention, which normal cells activate regularly with apoptotic/necrotic discard of miss-behaving cells. But stress on the replication S-period (Figure 1(B), wavy X&Y lines) with cause of slowed/ stalled replication forks, have shown completions of the cell cycle without activation of these check-point controls [76]. For the X-line with early S-period stress (likely DNA damage) this would lead to chromatin condensing G2 cells, being at the border of G1-phase, because of skip of mitosis, but unable to continue cell cycling from chromatin status in disagreement with that normally expected from telophase de-condensation (unpub). Our early work showed that the eventual continued cell cycling with 2n re-replication in second S-period gave rise to 4n cells with recombination chiasma between the 4-chromatids of diplochromosomes, a tritiated thymidine incorporation demonstration [30]. This work was corroborated one year later by a textbook “perfect picture” [31] [77] with excessive chiasma counts in Crohn’s syndrome with 46 numbered 4-chromatid chromosomes [31].

In an article from the Pellman laboratory [78], abnormal mitosis was linked to gain of DNA damage, which earlier had been shown to involve tetraploidization from spindle poison treated cells [79]. They presented data showing mitotic poison associated “prolonged mitosis”-, giving rise to 4n cells with mild mutational changes, a little later confirmed by different cancer-drug inductions. But the question of why non-DNA-damaging spindle poison produced mutations became an unsolved issue. However, considering that metaphase arrested diploid cells spend various times arrested (hours 6 - 16, depending on experiment) before there was observed “slip-back” into the cell cycle of the 2n cells with tetraploid cell-result from a second S-period. There would then have been various S-period stress periods with slowed-down replication cycles, affecting mutational changes in late replicating satellite DNAs. The consequent repair processes would be a source for genomic instability [10] [11]. These observations were verified from repair DNA foci, γ-H2AX, notably, in cancer cell lines [19] [20]. An explanation for this spindle poison-associated mutational change was offered by the suggestion that the p53 gene with the ability to “sense” mitotic time duration had become dysfunctional and had let diploid poison arrested cells slip through the G1/S checkpoint [80], and there is where it stands today. In general, the various types of producing polyploid cells are not considered for associated mutational happenings. But perhaps worse is the still use of spindle poisons in cancer treatment, which from above is a risky procedure for induction of relapse tumors with mutational changes.

Slowed-down S-periods, cell cycle compensated by incomplete replication of late replicating DNAs, as mentioned the non-coding satellite DNAs from nucleotide repetitive regions, which has four different chromosomal locations, the fragile sites, microsatellites, centromere and telomere regions [4] [5] [6] [19]. Repair of such lesions were early shown to involve multiple trials “within confined nucleotide regions” [81]. These trials were more recently shown by large RNA transcripts to “unite” fragile site repair locus with coding gene mutational happening located on the “border” of such unstable “dark DNA” repair [82]. Supporting is an earlier article also with focus on time prolonged cell cycle, “DNA replication stress underlies DNA, DSB (double strand breaks) formations in human precancerous lesions” [20]. In Figure 1, the Y line depicts late S-period stress, which is likely to produce less S-period, time disturbance with milder dark DNA mutational types (C > G, etc. types) which would not give rise to mitotic slippage process. Thus such cells would divide normally to proliferative diploid 2n/2C cells, likely not expressing fitness-gain with proliferative rate no different from normal cells. These diploid cells (First Cells)from late S-period stress we propose to be the origin of diploid cancers (48%of total tumor burden), which are “lazy proliferative cells” with decades to cancer phenotype [83].

The Glover laboratory, has spent a life time on experimental inductions on mutational events in dark-DNA, especially on how it can affect repair instability in common fragile sites (over 100 genomic distributed). They early-on showed that these “fragile sites” (FRA) were co-located with natural chromosomal structural more or less, weak break sites, which adds to the observed repair instability. Lately they used mild X-ray induced genome damage in normal cells, and observed molecular copy number alteration/variability (CNA/V), which has mutational inactivation affect. This tumorigenesis increasing finding is cancer-visually observed from frequent HSRs and DMs, in different types of cancers, but generally, lacking acceptance of gene-inactivation properties [10] [11]. Lastly, cancer therapy stands to gain important information from knowledge of how giant polyploid cells segregate into multinuclear cells, the PGCC [84] [85] [86] [87] [88], which also should be considered of being therapy resistant from surrounded extracellular matrix (See Wikipedia). In the past this latter hiding mechanism from cancer killing drugs had attention, which likely can be brought forward from the more advanced technology, which like-wise extended to the nuclear/cell budding process [21] [22] [23] [24] [25] could stop the PGCC from having tumor-regenerating capacity. In other words it is not enough to show/ suggest how these dangerous cells are generated, unless it is informative on today’s possible ways of therapy, remember it is a disease, not a mind “pleasing” biological problem.

The early mentioned association between satellite DNA repair instability and mutations in bordering located coding genes [4] [5] [6], evoked the question of whether the most frequently mutated genes among the SMGs would be chromosomally located in such “bad” chromosomal locations. For that inquiry the genes, TP53, Rb, CDKN2A, APC, RAS, MYC-N, MYC-C, MET and FHIT were found with the respective locations: 17p11.2, 13q13, 9p21.2, 5q22, 11p15, 2p24, 8q24, 7q21-31, 3p14.2 which surprisingly were all chromosomal sites near-by common fragile sites (FRAs). This was determined from G-banded genomes, which from Giemsa stain produce a white and black striped chromosomal reproducible pattern. Early-on these patterns were shown to achieve a most remarkable happening that of coding genes in thick and thin, black bands, whereas the white bands were almost devoid of such important genes (Therm [31]. In the summarized articles on common fragile sites [4] [5] more than 100 fragile sites are mapped to a haploid chromosomal genome with locations, which can be used to grossly assess where a gene is located, in dark bands or on their edges to white bands. Improved resolution can be achieved from ISCN Nomenclature [89], which shows G-banding of much longer chromosomes than generally done for karyotyping. For example, the CDKN2A gene (p16ink4a) is in a dark band on regular karyotype chromosomes, which became split into two dark bands by a white narrow band with the gene’ location 9p21.2, clearly seen on extended length chromosomes. Interestingly, p53 mutation being the most frequently mutated gene in tumors is located on chromosome #17p-arm on the border to centric heterochromatin, which give this gene several ways for mutational occur-rence: 1) from natural mutation rate occurrence, 2) from repair instability of either an under-replicated lesion or a natural replication error in the centric repetitive DNA, and 3) simple breakage with p-arm, p53-gene loss. Below is another list of both LOHs and single nucleotide mutational changes with fragile site relationships from various cancer-types (prostate, skin, lung) including a 1996 report from LOHs in breast and prostate cancers [90].

Some of these LOH loci were reported in more than one tumor similar to LOHs in breast hyper-plasia, discussed earlier (Wa 16, 17, 56). And interestingly, chromosome arms, 10p, 12p, 16p, 17p, 18p, 19p&q, 20q, 21q have no reported fragile sites in the 2007 map (D/G (4)), which rumors says has been renewed with more locations. It is a pity that the involved listed genes for these identified “bad” locations were not identified, but in the next instalment from melanoma cancer the list will include location, names of genes and their functions (unpub). The most important disclosure is that most of the LOHs were from deficiencies of nucleotides in heterozygous conditions, which explains genome instability and gene inactivation from CNA/V and their gross expressions HSRs & DMs (homogeneously staining regions and double minutes), frequently observed in cancer evolution. To this source for gene inactivation happenings there is also the fact that LOHs often express haplo-insufficiency or UPD (uniparental disomy), which all support our contention of loss of genetic function (gene inactivation) being a neutral/passive tumorigenesis evolutionary route to the mature cancer pheno-genotype. But from the list of LOHs in fragile sites the conclusion is that SMGs are not tumor selected-for,but are conse-quence from bad chromosomal locations near repair-unstable late replicating repetitive DNAs (dark DNA). This statement was convincingly shown to be true for the FHIT gene [4] [91] [92] [93]. A therapy relevant question in regard to these happenings, would likely focus on prevention of the dark-DNA repair processes [94], an approach which has already shown therapy success from new approved drug therapies. But in all of this conventional therapy works, there is a growing shift into algorithmic modeling of tumorigenic events, giving predictions of therapy vulnerability cancer mutational targets. The latest such algorithm is against key transcription factors in indivi-dualized, patient’s tumor [95]. But interestingly, the inventor (A. Califano) of this algorithmic approach in the commentary article says: “Theres’s say 1000 genes that are recurrently mutated across all tumors that may drive cancer” (our marking). These transcription regulators affects complicated interacting gene network-pathways controlled by genes being on or off, which is presented in a diagram of how a single drug targeting a master regulator, would stop the cancerous proliferative process. Our question is whether this algorithmic model would be therapy efficacy affected by tumor driver mutations not being selected-for, and giving hit and miss therapy results? But sadly, also in this model system as in other models, personal money gain is anticipated in the end of patient exploratory investigations, by the now familiar prior arrangement of for-profit (commercial) companies [8] [9] [12] [96]. An earlier commentator article told of CRISPR editing technology in exploratory work using volunteering deadly cancer-ill patients, which without demonstrated immediate side effects was considered a success with further larger trials being planned, the dying of the patients anyhow, apparently meant nothing. Explorative studies on the back of dying patients is neither ethical nor humane, even though papers for agreements were signed. Such desperate people should have high-priced counseling for risk and benefit. Where is that budget coming from? It is not only paradoxical, but plain sickening that arrangement for anticipated personal profit by sale of the used CRISPR methodology, already was arranged by establishment of a new commercial company [96]. When will we learn that “cancer for profit” decided back in the 1980^th, has devastating, cancer-negative effect. The greedy human mind [8] [9] although, competitive stimulating for new innovative ideas, it also adds to the cancer, financial industrial Goliath [12], untouchable, too big to fail. Nowhere in its existence would eradication of cancer be a desirable goal, and why fix something not yet broken? No wonder there is a rumor among scientists that cancer will be made into a liveable disease from a pill. Thus, the present high probability of cell-based immune competence for antibody response from the GR4n-SDS or their fitness derived First Cells with significant in vivo support [31] [39] [40] [41] [42] [43] [48] for vaccine explorations, will likely, never be a serious, open undertaking. The hope is that a commercial company decides to become much richer. But best is that a non-for profit organization becomes established for “out of the frame-type” cancer investigative data with offerings of further investigative, funded programs. This idea, especially directed to offerings of Ph.D. programs, exciting to the young, open clean mind, very likely can be a philanthropy giving, reaching the whole world (unpb.). However, only possible if the Will, is there.